Method of treating fibroproliferative disorders

ABSTRACT

Materials and methods for reducing cell proliferation or extracellular matrix production in a mammal are disclosed. The methods comprise administering to a mammal a composition comprising a therapeutically effective amount of a zvegf4 antagonist in combination with a pharmaceutically acceptable delivery vehicle. Exemplary zvegf4 antagonists include anti-zvegf4 antibodies, inhibitory polynucleotides, inhibitors of zvegf4 activation, and mitogenically inactive, receptor-binding variants of zvegf4. The materials and methods are useful in the treatment of, inter alia, fibroproliferative disorders of the kidney, liver, and bone.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of copendingapplication Ser. No. 09/564,595, filed May 3, 2000, and claims thebenefit of provisional applications Serial No. 60/132,250, filed May 3,1999, Serial No. 60/164,463, filed Nov. 10, 1999, Serial No. 60/180,169,filed Feb. 4, 2000, and No. 60/235,295, filed Sep. 26, 2000.

BACKGROUND OF THE INVENTION

[0002] Fibroproliferative disorders are characterized by the abnormalaccumulation of fibrous tissue (“fibrosis”) that can occur as a part ofthe wound-healing process in damaged tissue. Such tissue damage mayresult from physical injury, inflammation, infection, exposure totoxins, and other causes. The fibroproliferative condition includes botha cell growth component and an extensive phase characterized byextracellular matrix accumulation. Examples of fibroproliferativedisorders include dermal scar formation, keloids, liver fibrosis, lungfibrosis (e.g., silicosis, asbestosis), kidney fibrosis (includingdiabetic nephropathy), and glomerulosclerosis.

[0003] A variety of renal diseases can be classified asfibroproliferative. Glomerular (usually mesangial) cell proliferationoccurs in many types of glomerulonephritides in conjunction withincreased extracellular matrix accumulation (Iida et al., Proc. Natl.Acad. Sci. USA 88:6560-6564, 1991). For example, mesangial cellproliferation precedes glomerulosclerosis in the remnant kidney model(Floege et al., Kidney International 41:297-309, 1992), and experimentaloverexpression of growth factors such as PDGF-B and TGF-beta in thekidney induces cell proliferation, matrix accumulation, andglomerulosclerosis (Isaka et al., J. Clin. Invest. 92:2597-2601, 1993;Cybulsky, Curr. Opin. Nephropathy and Hypert. 9:217-223, 2000).

[0004] A number of vascular pathologies result from a combination ofmesenchymal cell proliferation (smooth muscle and fibroblast-like) andextensive accumulation of extracellular matrix components. Such arterywall diseases as arteriosclerotic lesions, arteritis of various origins,and the vascular re-stenotic lesions that frequently follow angioplasty(Riessen et al., Am. Heart J. 135:357-364, 1998; Plenz et al.,Arterioscler. Thromb. Vase. Biol. 17:2489-2499, 1997; McCaffrey,Cytokine Growth Factor Rev. 11:103-114, 2000) are consideredfibroproliferative. Other fibroproliferative responses include thefiborproliferative responses that occur in organs following transplant(e.g., heart transplants), at sites of vascular anastamosis, and atareas around catheter placements (e.g., arterio-venous shunts used fordialysis).

[0005] Bone formation, both physiologic and pathologic, can be describedas the interplay between bone formation that results from proliferationof osteoblasts and production by them of extracellular matrix, and thereplication of osteoclasts and their modulation of this matrix. Diseaseswhere there is aberrant and ectopic bone formation, such as thatoccurring with prostate tumor metastases to the axial skeleton, arecommonly characterized by active proliferation of the major cell typesparticipating in bone formation as well as by elaboration by them of acomplex bone matrix. These diseases can therefore be viewed asfibroproliferative.

[0006] Pulmonary fibrosis is a major cause of morbidity and mortality.Pulmonary fibrosis is associated with the use of high-doseantineoplastic agents (e.g., bleomycin) in chemotherapy and with bonemarrow transplantation for cancer treatment. The development of lungdisease is the major dose-limiting side effect of bleomycin. See, Tranet al., J. Clin. Invest. 99:608-617, 1997. Idiopathic pulmonary fibrosis(IPF) is another lung fibrotic disease characterized by afibroproliferative response. Various factors, including aspiration andexposure to environmental pollutants may result in IPF (Egan, The Lancet354:1839-1840, 1999). The standard treatment for IPF is oralglucocorticoids. However, lung function improves in less than 30 percentof patients who receive this treatment, and, regardless of treatment,the median survival is four to five years after the onset of symptoms.The proliferation of fibroblasts and the accumulation of interstitialcollagens are the hallmarks of progressive organ fibrosis, however thebiochemical mechanism of induction of lung fibrosis remains unclear(Ziesche et al., New Eng. J. Med. 341:1264-1269, 1999; Kuwano et al., J.Clin Invest. 104:13-19, 1999). Pulmonary hypertension results from avariety of initiating stimuli. Its progression is associated withpulmonary vascular sclerosis, which includes abnormal endothelialmorphology and function, muscularization of normally nonmuscularperipheral arteries related to differentiation of pericytes, and medialhypertrophy and neointimal formation in muscular arteries as aconsequence of hypertrophy, proliferation, and migration of residentsmooth muscle cells and increased production of extracellular matrixcomponents. These components include collagen, elastin, fibronectin, andtenascin-C. This fibroproliferative response can progress tolife-threatening pulmonary arterial obstructive disease (Cowan et al.,J. Clin. Invest. 105:21-34, 2000).

[0007] Liver (hepatic) fibrosis occurs as a part of the wound-healingresponse to chronic liver injury. Fibrosis occurs as a complication ofhaemochromatosis, Wilson's disease, alcoholism, schistosomiasis, viralhepatitis, bile duct obstruction, toxin exposure, and matabolicdisorders. This formation of scar tissue is believed to represent anattempt by the body to encapsulate the injured tissue. Liver fibrosis ischaracterized by the accumulation of extracellular matrix that can bedistinguished qualitatively from that in normal liver. Left unchecked,hepatic fibrosis progresses to cirrhosis (defined by the presence ofencapsulated nodules), liver failure, and death.

[0008] In recent years there have been significant advances in theunderstanding of the cellular and biochemical mechanisms underlyingliver fibrosis (reviewed by Li and Friedman, J. Gastroenterol. Hepatol.14:618-633, 1999). Stellate (Ito) cells are believed to be a majorsource of extracellular matrix in the liver. Stellate cells respond to avariety of cytokines present in the liver, some of which they alsoproduce (Friedman, Seminars in Liver Disease 19:129-140, 1999).

[0009] As summarized by Li and Friedman (ibid.), actual and proposedtherapeutic strategies for liver fibrosis include removal of theunderlying cause (e.g., toxin or infectious agent), suppression ofinflammation (using, e.g., corticosteroids, IL-1 receptor antagonists,or other agents), down-regulation of stellate cell activation (using,e.g., gamma interferon or antioxidants), promotion of matrixdegradation, or promotion of stellate cell apoptosis. Despite recentprogress, many of these strategies are still in the experimental stage,and existing therapies are aimed at suppressing inflammation rather thanaddressing the underlying biochemical processes. Thus, there remains aneed in the art for materials and methods for treatingfibroproliferative disorders, including liver fibrosis.

DESCRIPTION OF THE INVENTION

[0010] Within one aspect of the present invention there is provided amethod of reducing proliferation of or extracellular matrix productionby a cell in a mammal comprising administering to the mammal acomposition comprising a therapeutically effective amount of a zvegf4antagonist in combination with a pharmaceutically acceptable deliveryvehicle, wherein the zvegf4 antagonist is selected from the groupconsisting of anti-zvegf4 antibodies, inhibitory polynucleotides,inhibitors of zvegf4 activation, and mitogenically inactive,receptor-binding variants of zvegf4. Within certain embodiments of theinvention the proliferation of mesangial, epithelial, endothelial,smooth muscle, fibroblast, osteoblast, osteoclast, neuronal, stromal,stellate, or interstitial cells is reduced. Within another embodiment ofthe invention the proliferation of tumor cells, such as prostate tumorcells, is reduced. Within another embodiment of the inventionextracellular matrix production is reduced. Within other embodiments ofthe invention the mammal is suffering from a fibroproliferative disorderof the kidney, liver, or bone.

[0011] Within a related aspect of the invention there is provided amethod of reducing proliferation of or extracellular matrix productionby a cell in a mammal, wherein the cell is an epithelial, endothelial,smooth muscle, fibroblast, osteoblast, neuronal, or stellate cell, themethod comprising administering to the mammal a composition comprising atherapeutically effective amount of a zvegf4 antagonist in combinationwith a pharmaceutically acceptable delivery vehicle, wherein the zvegf4antagonist is selected from the group consisting of anti-zvegf4antibodies, inhibitory polynucleotides, inhibitors of zvegf4 activation,and mitogenically inactive, receptor-binding variants of zvegf4.

[0012] Within a further aspect of the invention there is provided amethod of reducing proliferation of or extracellular matrix productionby prostate tumor cells in a mammal, the method comprising administeringto the mammal a composition comprising a therapeutically effectiveamount of a zvegf4 antagonist in combination with a pharmaceuticallyacceptable delivery vehicle, wherein the zvegf4 antagonist is selectedfrom the group consisting of anti-zvegf4 antibodies, inhibitorypolynucleotides, inhibitors of zvegf4 activation, and mitogenicallyinactive, receptor-binding variants of zvegf4.

[0013] Within another aspect of the invention there is provided a methodof reducing metastasis of prostate cancer cells to bone in a mammal, themethod comprising administering to the mammal a composition comprising atherapeutically effective amount of a zvegf4 antagonist in combinationwith a pharmaceutically acceptable delivery vehicle, wherein the zvegf4antagonist is selected from the group consisting of anti-zvegf4antibodies, inhibitory polynucleotides, inhibitors of zvegf4 activation,and mitogenically inactive, receptor-binding variants of zvegf4.

[0014] Within a further aspect of the invention there is provided amethod of treating a fibroproliferative disorder in a mammal comprisingadministering to the mammal a composition comprising a therapeuticallyeffective amount of a zvegf4 antagonist in combination with apharmaceutically acceptable delivery vehicle, wherein the zvegf4antagonist is selected from the group consisting of anti-zvegf4antibodies, inhibitors of zvegf4 activation, mitogenically inactivereceptor-binding zvegf4 variant polypeptides, and inhibitorypolynucleotides. Within certain embodiments of the invention thefibroproliferative disorder is a fibroproliferative disorder of liver,kidney, or bone.

[0015] Within an additional aspect of the invention there is provided amethod of reducing stellate cell activation in a mammal comprisingadministering to the mammal a composition comprising a zvegf4 antagonistin combination with a pharmaceutically acceptable delivery vehicle,wherein the zvegf4 antagonist is selected from the group consisting ofanti-zvegf4 antibodies, mitogenically inactive receptor-binding zvegf4variant polypeptides, and inhibitory polynucleotides, in an amountsufficient to reduce stellate cell activation.

[0016] Within certain embodiments of the above-disclosed methods, thezvegf4 antagonist is selected from the group consisting of anti-zvegf4antibodies and inhibitory polynucleotides. Within other embodiments, thezvegf4 antagonist is an anti-zvegf4 antibody. Within additionalembodiments of these methods, the zvegf4 antagonist is administered incombination with an antagonist of a second growth factor.

[0017] These and other aspects of the invention will become evident uponreference to the following detailed description of the invention and theaccompanying FIGURE.

[0018] The FIGURE is a Hopp/Woods hydrophilicity profile of the aminoacid sequence shown in SEQ ID NO:2. The profile is based on a slidingsix-residue window. Buried G, S, and T residues and exposed H, Y, and Wresidues were ignored. These residues are indicated in the FIGURE bylower case letters.

[0019] The term “antagonist” is used herein to denote a compound thatreduces a biological activity of another compound. Within the presentinvention, a “zvegf4 antagonist” is a compound that reduces thereceptor-mediated biological activity (e.g., mitogenic activity) ofzvegf4 on a target cell. Antagonists may exert their action by competingwith zvegf4 for binding sites on a cell-surface receptor, by binding tozvegf4 and preventing it from binding to a cell-surface receptor, byotherwise interfering with receptor function, by reducing production ofzvegf4, or by other means.

[0020] “Extracellular matrix” (ECM) is a complex mixture ofmacromolecules that accumulates within tissues in close apposition tocell surfaces. ECM contains secreted macromolecules such as collagens I,III and IV; fibronectin; laminins; and various proteoglycans. Thesemacromolecules can be organized to provide cohesion to the tissue andcan contribute to its structural and mechanical properties. ECM can actas a depository for, and release site of, potent secreted growthfactors, and is known to influence growth, survival and differentiationof the cells it surrounds. Pathologic ECM accumulation, if unchecked,can restrict access of nutrients, growth factors, and otherphysiologically important molecules to cells and can lead to thecreation of areas of low live cell density. Over time, this accumulationcan result in the inability of a tissue to perform its specificmetabolic and structural roles, and may ultimately lead to overt celland tissue death.

[0021] An “inhibitory polynucleotide” is a DNA or RNA molecule thatreduces or prevents expression (transcription or translation) of asecond (target) polynucleotide. Inhibitory polynucleotides includeantisense polynucleotides, ribozymes, and external guide sequences. Theterm “inhibitory polynucleotide” further includes DNA and RNA moleculesthat encode the actual inhibitory species, such as DNA molecules thatencode ribozymes.

[0022] The terms “treat” and “treatment” are used broadly to denotetherapeutic and prophylactic interventions that favorably alter apathological state. Treatments include procedures that moderate orreverse the progression of, reduce the severity of, prevent, or cure adisease.

[0023] The term “zvegf4 protein” is used herein to denote proteinscomprising the growth factor domain of a zvegf4 polypeptide (e.g.,residues 258-370 of human zvegf4 (SEQ ID NO:2) or mouse zvegf4 (SEQ IDNO:4)), wherein the protein is mitogenic for cells expressingcell-surface PDGF α- and/or β-receptor subunit. Zvegf4 has been found toactivate the αα, αβ, and ββ isoforms of PDGF receptor. Zvegf4 proteinsinclude homodimers and heterodimers as disclosed below. Using methodsknown in the art, zvegf4 proteins can be prepared in a variety of forms,including glycosylated or non-glycosylated, pegylated or non-pegylated,with or without an initial methionine residue, and as fusion proteins asdisclosed in more detail below.

[0024] All references cited herein are incorporated by reference intheir entirety.

[0025] The present invention provides methods for reducing proliferationof or extracellular matrix production by a cell in a mammal using zvegf4antagonists. The invention further provides methods of treatingfibroproliferative disorders in a mammal using zvegf4 antagonists.Zvegf4 is a multi-domain protein that is structurally related toplatelet-derived growth factor (PDGF) and the vascular endothelialgrowth factors (VEGF). This protein is also referred to as “PDGF-D”(WIPO Publication WO 00/27879).

[0026] Structural predictions based on the zvegf4 sequence and itshomology to other growth factors suggests that the polypeptide can formhomomultimers or heteromultimers that act on tissues by modulating cellproliferation, migration, differentiation, or metabolism. Experimentalevidence supports these predictions. Zvegf4 heteromultimers may comprisea polypeptide from another member of the PDGF/VEGF family of proteins,including VEGF, VEGF-B, VEGF-C, VEGF-D, zvegf3/PDGF-C (WO 00/34474),PlGF (Maglione et al., Proc. Natl. Acad. Sci. USA 88:9267-9271, 1991),PDGF-A (Murray et al., U.S. Pat. No. 4,899,919; Heldin et al., U.S. Pat.No. 5,219,759), or PDGF-B (Chiu et al., Cell 37:123-129, 1984; Johnssonet al., EMBO J. 3:921-928, 1984).

[0027] The zvegf4 polypeptide chain comprises a growth factor domain, aCUB domain, and an interdomain linking the CUB and growth factordomains. The growth factor domain is characterized by an arrangement ofcysteine residues and beta strands that is characteristic of the“cystine knot” structure of the PDGF family. The CUB domain showssequence homology to CUB domains in the neuropilins (Takagi et al.,Neuron 7:295-307, 1991; Soker et al., Cell 92:735-745, 1998), human bonemorphogenetic protein-i (Wozney et al., Science 242:1528-1534, 1988),porcine seminal plasma protein and bovine acidic seminal fluid protein(Romero et al., Nat. Struct. Biol. 4:783-788, 1997), and X. laevistolloid-like protein (Lin et al., Dev. Growth Differ. 39:43-51, 1997).

[0028] A representative human zvegf4 polypeptide sequence is shown inSEQ ID NO:2, and a representative mouse zvegf4 polypeptide sequence isshown in SEQ ID NO:4. DNAs encoding these polypeptides are shown in SEQID NOS:1 and 3, respectively. Analysis of the amino acid sequence shownin SEQ ID NO:2 indicates that residues 1 to 18 form a secretory peptide.The CUB domain extends from residue 52 to residue 179. A propeptide-likesequence extends from residue 180 to either residue 245, residue 249 orresidue 257, and includes four potential cleavage sites at its carboxylterminus, monobasic sites at residue 245 and residue 249, a dibasic siteat residues 254-255, and a target site for furin or a furin-likeprotease at residues 254-257. Protein produced in a baculovirusexpression system showed cleavage between residues 249 and 250, andincluded longer species with amino termini at residues 19 and 35. Thegrowth factor domain extends from residue 258 to residue 370, and mayinclude additional residues at the N-terminus (e.g., residues 250 to 257or residues 246 to 257). Those skilled in the art will recognize thatdomain boundaries are somewhat imprecise and can be expected to vary byup to ±5 residues from the specified positions. Corresponding domains inmouse and other non-human zvegf4s can be determined by those of ordinaryskill in the art from sequence alignments. Cleavage of full-length humanzvegf4 with plasmin resulted in activation of the zvegf4 polypeptide asdetermined in a cellular assay using a PDGF receptor and luciferasereporter gene. By Western analysis, a band migrating at approximatelythe same size as the growth factor domain was observed in thispreparation.

[0029] Signal peptide cleavage is predicted to occur in human zvegf4after residue 18 (±3 residues). Upon comparison of human and mousezvegf4 sequences, alternative signal peptide cleavage sites arepredicted after residue 23 and/or residue 24. This analysis suggeststhat the zvegf4 polypeptide chain may be cleaved to produce a pluralityof monomeric species, some of which are shown in Table 1. In certainhost cells, cleavage after Lys-255 is expected to result in subsequentremoval of residues 254-255, although polypeptides with a carboxylterminus at residue 255 may also be prepared. Cleavage after Lys-257 isexpected to result in subsequent removal of residue 257. These cleavagesites can be modified to prevent proteolysis and thus provide for theproduction of uncleaved zvegf4 polypeptides and multimers comprisingthem. Actual cleavage patterns are expected to vary among host cells.TABLE 1 Monomer Residues (SEQ ID NO:2) CUB domain 19-179 24-179 25-17935-179 52-179 CUB domain + interdomain region 19-257 24-257 25-25735-257 52-257 19-255 24-255 25-255 35-255 52-255 19-253 24-253 25-25335-253 52-253 19-249 24-249 25-249 35-249 52-249 19-245 24-245 25-24535-245 52-245 CUB domain + interdomain region + 19-370 growth factordomain 24-370 25-370 35-370 52-370 Growth factor domain 246-370 250-370  258-370  Growth factor domain + 180-370  interdomain region

[0030] Zvegf4 can thus be prepared in a variety of multimeric formscomprising a zvegf4 polypeptide as disclosed above. These zvegf4polypeptides include zvegf4₁₉₋₃₇₀, zvegf4₅₂₋₃₇₀, zvegf4₂₄₆₋₃₇₀,zvegf4₂₅₀₋₃₇₀, and zvegf4₂₅₈₋₃₇₀. Variants and derivatives of thesepolypeptides can also be prepared as disclosed herein.

[0031] Zvegf4 proteins can be prepared as fusion proteins comprisingamino- or carboxyl-terminal extensions, such as an amino-terminalmethionine residue, an affinity tag, or a targetting polypeptide. Forexample, a zvegf4 protein can be prepared as a fusion with an affinitytag to facilitate purification. In principal, any peptide or protein forwhich an antibody or other specific binding agent is available can beused as an affinity tag. Affinity tags include, for example, apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-1210,1988), streptavidin binding peptide, maltose binding protein (Guan etal., Gene 67:21-30, 1987), cellulose binding protein, thioredoxin,ubiquitin, T7 polymerase, or other antigenic epitope or binding domain.Fusion of zvegf4 to, for example, maltose binding protein or glutationeS transferase can be used to improve yield in bacterial expressionsystems. In these instances the non-zvegf4 portion of the fusion proteinordinarily will be removed prior to use. Separation of the zvegf4 andnon-zvegf4 portions of the fusion protein is facilitated by providing aspecific cleavage site between the two portions. Such methods are wellknown in the art. Zvegf4 can also be fused to a targetting peptide, suchas an antibody (including polyclonal antibodies, monoclonal antibodies,antigen-binding fragments thereof such as F(ab′)₂ and Fab fragments,single chain antibodies, and the like) or other peptidic moiety thatbinds to a target tissue.

[0032] Variations can be made in the zvegf4 amino acid sequences shownin SEQ ID NO:2 and SEQ ID NO:4 to provide inactive, receptor-bindingpolypeptides that act as zvegf4 antagonists. Such variations includeamino acid substitutions, deletions, and insertions. While not wishingto be bound by theory, it is believed that residues within regions273-295 and 307-317 of human zveg4 (SEQ ID NO:2) may be involved inligand-receptor interactions. It is also believed that the CUB domainmay mediate the binding of zvegf4 to certain cell-surface receptors,thereby providing a targeting function for delivery of the growth factordomain. The CUB domain, in the absence of an active growth factordomain, may therefore be useful as a zvegf4 antagonist. The effects ofamino acid sequence changes at specific positions in zvegf4 proteins canbe assessed using procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA88:4498-4502, 1991). Multiple amino acid substitutions can be made andtested using known methods of mutagenesis and screening, such as thosedisclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowieand Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Other methodsthat can be used include phage display (e.g., Lowman et al., Biochem.30:10832-10837, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPOPublication WO 92/06204), region-directed mutagenesis (Derbyshire etal., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988), and DNA shuffling(Stemmer, Nature 370:389-391, 1994; Stemmer, Proc. Natl. Acad. Sci. USA91:10747-10751, 1994). The resultant mutant molecules are tested forreceptor binding, mitogenic activity, or other properties (e.g.,stimulation of extracellular matrix production) to identify amino acidresidues that are critical to these functions. Mutagenesis can becombined with high-volume or high-throughput screening methods to detectbiological activity of zvegf4 variant polypeptides, including biologicalactivity in modulating cell proliferation. For example, mitogenesisassays that measure dye incorporation or ³H-thymidine incorporation canbe carried out on large numbers of samples. Competition assays can beemployed to confirm antagonist activity.

[0033] Zvegf4 proteins, including full-length proteins, variant proteins(including antagonists), biologically active fragments, and fusionproteins, can be produced in genetically engineered host cells accordingto conventional techniques. Suitable host cells are those cell typesthat can be transformed or transfected with exogenous DNA and grown inculture, and include bacteria, fungal cells, and cultured highereukaryotic cells (including cultured cells of multicellular organisms).Techniques for manipulating cloned DNA molecules and introducingexogenous DNA into a variety of host cells are disclosed by Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al.,eds., Current Protocols in Molecular Biology, Green and Wiley and Sons,NY, 1993. In general, a DNA sequence encoding a zvegf4 polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers,vectors, and other elements is a matter of routine design within thelevel of ordinary skill in the art. Many such elements are described inthe literature and are available through commercial suppliers. See, forexample, WO 00/34474.

[0034] Zvegf4 proteins can comprise non-naturally occurring amino acidresidues. Non-naturally occurring amino acids include, withoutlimitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are knownin the art for incorporating non-naturally occurring amino acid residuesinto proteins. For example, an in vitro system can be employed whereinnonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations is carried out in a cell-freesystem comprising an E. coli S30 extract and commercially availableenzymes and other reagents. Proteins are purified by chromatography.See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991;Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science259:806-809, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA90:10145-10149, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-19998, 1996). Within a third method, E. coli cells arecultured in the absence of a natural amino acid that is to be replaced(e.g., phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

[0035] Zvegf4 polypeptides or fragments thereof can also be preparedthrough chemical synthesis according to methods known in the art,including exclusive solid phase synthesis, partial solid phase methods,fragment condensation or classical solution synthesis. See, for example,Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid PhasePeptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, Ill.,1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford,1989.

[0036] Zvegf4 proteins are purified by conventional protein purificationmethods, typically by a combination of chromatographic techniques. See,in general, Affinity Chromatography: Principles & Methods, Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York, 1994. Proteinscomprising a polyhistidine affinity tag (typically about 6 histidineresidues) are purified by affinity chromatography on a nickel chelateresin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325,1988. Proteins comprising a glu-glu tag can be purified byimmunoaffinity chromatography according to conventional procedures. See,for example, Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4,1985. Maltose binding protein fusions are purified on an amylose columnaccording to methods known in the art. Zvegf4 growth factor domainprotein can be purified using a combination of chromatography on astrong cation exchanger followed by affinity chromatography andsize-exclusion chromatography.

[0037] As shown in more detail in the examples that follow, zvegf4 ishighly expressed in the kidney, and over-expression of zvegf4 in mice byinjection of an adenovirus vector encoding zvegf4 elicitsfibroproliferative changes in the kidney. Two readily identifiablefeatures of these changes are (a) enlarged glomeruli due in part tomesangial cell proliferation, and (b) tubular regeneration due to tubuleepithelial cell proliferation. These findings indicate that an increasein zvegf4 protein can modify the function of, and the interactionsamong, mesangial, epithelial, endothelial, smooth muscle, andinterstitial cells, which are all key players in glomerular and vasculardiseases of the kidney. Furthermore, zvegf4 has been found to affectcell proliferation in at least some of these cells in vitro. Experimentshave also shown that the activity of zvegf4 is mediated by two PDGFreceptor subunits, alpha and beta (PDGF-αR and PDGF-βR). These receptorsubunits are widely is expressed in most renal cell types, and theirexpression is upregulated in a number of kidney pathologies (e.g., Iidaet al., Proc. Natl. Acad. Sci. USA 88:6560-6564, 1991). Stimulation ofPDGF receptors has been implicated in fibroproliferative diseases of thekidney in a variety of animal models (e.g., Ooi et al., P.S.E.B.M.213:230-237, 1996; Lindahl et al., Development 125:3313-3322, 1998;Lindahl and Betsholtz, Curr. Op. Nephr. Hypert. 7:21-26, 1998; andBetsholtz and Raines, Kidney Int. 51:1361-1369, 1997).

[0038] As also shown herein, zvegf4 has been found to stimulate theproduction of TGF-β by rat liver stellate cells. TGF-β is thought to bea major mediator of fibrosis, due to its ability to stimulateextracellular matrix synthesis (especially collagen and fibronectin) ina variety of mesenchymal cell types, most notably fibroblasts. TGF-β hasbeen implicated in the development of fibrosis of the heart, lung,liver, and kidney (Ledbetter et al., Kidney Int. 58(6):2367-2376, 2000;Chen et al., Mol. Cell Cardiol. 32(10):1805-1819, 2000; Nakamura et al.,Hepatology 32(2):247-255, 2000; Martin et al., Int. J. Radiat. Oncol.Biol. Phys. 47(2):277-290, 2000; Sanderson et al., Proc. Natl. Acad.Sci. USA 2(7):2572-2576, 1995). Increased expression of zvegf4 in organssuch as the heart, kidney, lung or liver may result in fibrosis, whichmay at least in part be mediated and exacerbated by the enhancedproduction of TGF-β.

[0039] Zvegf4 has been found to be highly expressed in mouse prostatetumor cell lines as shown by Northern blotting. In addition, animalstreated with a zvegf4-encoding adenovirus vector displayed invasion ofthe marrow space by endosteal bone, indicating an effect of zvegf4 onbone growth. In view of the high incidence of bony metastases in mensuffering from prostate cancer, these results implicate zvegf4 as amediator of prostate tumor-related cancellous bone growth.

[0040] Additional evidence indicates that zvegf4 may bind tocell-surface semaphorins, presumably via the CUB domain. Cells havingcell-surface semaphorins include endothelial cells, neuronal cells,lymphocytes, and various tumor cells.

[0041] In view of the experiments summarized above and disclosed in moredetail herein, it is expected that altered zvegf4 expression mayinitiate or exacerbate renal disease and other fibroproliferativedisorders. In this context, inhibiting the action of zvegf4 using azvegf4 antagonist will limit the progress of such disorders. Zvegf4antagonists include, without limitation, anti-zvegf4 antibodies(including neutralizing antibodies), soluble zvegf4 receptors (includingsoluble PDGF beta receptor; see, e.g., Herren et al., J. Biol. Chem.268:15088-15095, 1993; U.S. Pat. No. 6,018,026; and soluble PDGF alphareceptor), anti-receptor antibodies, and other peptidic and non-peptidicagents, including ribozymes, antisense polynucleotides, small moleculeinhibitors, and mitogenically inactive, receptor-binding zvegf4polypeptides.

[0042] Within the present invention zvegf4 antagonists are used to blockthe proliferative or profibrotic effects of zvegf4. Thus, the presentinvention provides methods of inhibiting, reducing, preventing, orotherwise treating fibroproliferative disorders, including, withoutlimitation, scar formation, keloids, scleroderma, liver fibrosis, lungfibrosis, kidney fibrosis, myelofibrosis, post-surgical fibroticadhesions, fibrotic tumors, fibroproliferative disorders of thevasculature, fibroproliferative disorders of the prostate,fibroproliferative disorders of bone, fibromatosis, fibroma,fibrosarcoma, and the like.

[0043] Fibroproliferative disorders of the kidney include, withoutlimitation, glomerulonephritis (including membranoproliferative, diffuseproliferative, rapidly progressive, and chronic forms), diabeticglomerulosclerosis, focal glomerulosclerosis, diabetic nephropathy,lupus nephritis, tubulointerstitial fibrosis, membranous nephropathy,amyloidosis (which affects the kidney among other tissues), renalarteriosclerosis, and nephrotic syndrome. The glomerulus is a majortarget of many types of renal injury, including immunologic (e.g.,immune-complex- or T-cell-mediated), hemodynamic (systemic or renalhypertension), metabolic (e.g., diabetes), “atherosclerotic”(accumulation of lipids in the glomerulus), infiltrative (e.g.,amyloid), and toxic (e.g., snake venom) injuries (Johnson, Kidney Int.45:1769-1782, 1994). The renal structural changes in patients withdiabetic nephropathy include hypertrophy of the glomerulus, thickeningof the glomerular and tubular membranes (due to accumulated matrix), andincreased amounts of matrix in the measangium and tubulointerstitium(Ziyadeh et al., Proc. Natl. Acad. Sci. USA 97:8015-8020, 2000).Glomerular hypertension due to intrarenal hemodynamic changes indiabetes can contribute to the progression of diabetic nephropathy(Ishida et al., Diabetes 48:595-602, 1999). Autoimmune nephritis canalso lead to altered mesangial cell growth responses (Liu and Ooi, J.Immunol. 151:2247-2251, 1993). Infection by hepatitis-C virus can alsoresult in idiopathic membranoproliferative glomerulonephritis (Johnsonet al., N. Engl. J. Med. 328:465-470, 1993).

[0044] Fibroproliferative disorders of the lung include, for example,silicosis, asbestosis, idiopathic pulmonary fibrosis, bronchiolitisobliterans-organizing pneumonia, pulmonary fibrosis associated withhigh-dose chemotherapy, idiopathic pulmonary fibrosis, and pulmonaryhypertension. These diseases are characterized by cell proliferation andincreased production of extracellular matrix components, such ascollagens, elastin, fibronectin, and tenascin-C.

[0045] Fibrosis of the liver can result from damage due to chronic liverdisease, including chronic active hepatitis (including hepatitis C) andmany other types of cirrhosis. Widespread, massive necrosis, includingdestruction of virtually the entire liver, can be caused by, inter alia,fulminant viral hepatitis; overdoses of the analgesic acetaminophen;exposure to other drugs and chemicals such as halothane, monoamineoxidase inhibitors, agents employed in the treatment of tuberculosis,phosphorus, carbon tetrachloride, and other industrial chemicals.Conditions associated with ultrastructural lesions that do notnecessarily produce obvious liver cell necrosis include Reye's syndromein children, tetracycline toxicity, and acute fatty liver of pregnancy.Cirrhosis, a diffuse process characterized by fibrosis and a conversionof normal architecture into structurally abnormal nodules, can comeabout for a variety reasons including alcohol abuse, post necroticcirrhosis (usually due to chronic active hepatitis), biliary cirrhosis,pigment cirrhosis, cryptogenic cirrhosis, Wilson's disease, andalpha-1-antitrypsin deficiency. In cases of liver fibrosis it may bebeneficial to administer a zvegf4 antagonist to suppress the activationof stellate cells, which have been implicated in the production ofextracellular matrix in fibrotic liver (Li and Friedman, J.Gastroenterol. Hepatol. 14:618-633, 1999).

[0046] Diseases of the skeleton that are due to modified growth andmatrix production in the bone include, but are not limited to,osteopetrosis, hyperostosis, osteosclerosis, osteoarthritis, and ectopicbone formation in metastatic prostate cancer. Fibroproliferativedisorders of bone are characterized by aberrant and ectopic boneformation, commonly seen as active proliferation of the major cell typesparticipating in bone formation as well as elaboration by those cells ofa complex bone matrix. Exemplary of such bone disorders is the fibrosisthat occurs with prostate tumor metastases to the axial skeleton. Inprostate tumor-related cancellous bone growth, prostate carcinoma cellscan interact reciprocally with osteoblasts to produce enhanced tumorgrowth and osteoblastic action when they are deposited in bone (Zhau etal., Cancer 88:2995-3001, 2000; Ritchie et al., Endocrinology138:1145-1150, 1997). As disclosed in more detail below, mice receivinga zvegf4-encoding adenovirus vector displayed a similar pathology asthat observed in prostate cancer patients who display tumor metastasesin the axial skeleton and consequent formation of endosteal bone. Inaddition, a panel of mouse prostate cell lines (epithelial and stromal)propagated in culture were found to express very high levels of zvegf4messenger RNA. These data suggest that zvegf4 is involved (via autocrineand/or paracrine mechanisms) in prostate tumor growth, metastasis, andeffects in bone. Fibroproliferative responses of the bone originating inthe skeleton per se include osteopetrosis and hyperstosis. A defect inosteoblast differentiation and function is thought to be a major causein osteopetrosis, an inherited disorder characterized by bone sclerosisdue to reduced bone resorption, wherein marrow cavities fail to develop,resulting in extramedullary hematopoiesis and severe hematologicabnormalities associated with optic atrophy, deafness, and mentalretardation (Lajeunesse et al., J. Clin Invest. 98:1835-1842, 1996). Inosteoarthritis, bone changes are known to occur, and bone collagenmetabolism is increased within osteoarthritic femoral heads. Thegreatest changes occur within the subchondral zone, supporting a greaterproportion of osteoid in the diseased tissue (Mansell and Bailey, J.Clin. Invest. 101:1596-1603, 1998).

[0047] Fibroproliferative disorders of the vasculature include, forexample, transplant vasculopathy, which is a major cause of chronicrejection of heart transplantation. Transplant vasculopathy ischaracterized by accelerated atherosclerotic plaque formation withdiffuse occlusion of the coronary arteries, which is a “classic”fibroproliferative disease. See, Miller et al., Circulation101:1598-1605, 2000).

[0048] Antibodies used as zvegf4 antagonists include antibodies thatspecifically bind to a zvegf4 protein or a zvegf4 cell-surface receptorand, by so binding, reduce or prevent the binding of zvegf4 protein tothe receptor and, consequently, reduce or block the receptor-mediatedactivity of zvegf4. As used herein, the term “antibodies” includespolyclonal antibodies, affinity-purified polyclonal antibodies,monoclonal antibodies, and antigen-binding fragments, such as F(ab′)₂and Fab proteolytic fragments. Genetically engineered intact antibodiesor fragments, such as chimeric antibodies, Fv fragments, single chainantibodies, and the like, as well as synthetic antigen-binding peptidesand polypeptides, are also included. Non-human antibodies may behumanized by grafting non-human CDRs onto human framework and constantregions, or by incorporating the entire non-human variable domains(optionally “cloaking” them with a human-like surface by replacement ofexposed residues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Monoclonal antibodies can also beproduced in mice that have been genetically altered to produceantibodies that have a human structure.

[0049] Methods for preparing and isolating polyclonal and monoclonalantibodies are well known in the art. See, for example, Cooligan et al.(eds.), Current Protocols in Immunology, National Institutes of Health,John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: ALaboratory Manual, second edition, Cold Spring Harbor, N.Y., 1989; andHurrell (ed.), Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press, Inc., Boca Raton, Fla., 1982. As would beevident to one of ordinary skill in the art, polyclonal antibodies canbe generated by inoculating a variety of warm-blooded animals such ashorses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats witha zvegf4 polypeptide or a fragment thereof.

[0050] Immunogenic polypeptides will comprise an epitope-bearing portionof a zvegf4 polypeptide (e.g., as shown in SEQ ID NO:2) or receptor. An“epitope” is a region of a protein to which an antibody can bind. See,for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002,1984. Epitopes can be linear or conformational, the latter beingcomposed of discontinuous regions of the protein that form an epitopeupon folding of the protein. Linear epitopes are generally at least 6amino acid residues in length. Relatively short synthetic peptides thatmimic part of a protein sequence are routinely capable of eliciting anantiserum that reacts with the partially mimicked protein. See,Sutcliffe et al., Science 219:660-666, 1983. Immunogenic,epitope-bearing polypeptides contain a sequence of at least six, oftenat least nine, more often from 15 to about 30 contiguous amino acidresidues of a zvegf4 protein or receptor. Polypeptides comprising alarger portion of a zvegf4 protein or receptor, i.e. from 30 to 50residues up to the entire sequence are included. It is preferred thatthe amino acid sequence of the epitope-bearing polypeptide is selectedto provide substantial solubility in aqueous solvents, that is thesequence includes relatively hydrophilic residues, and hydrophobicresidues are substantially avoided (see the FIGURE). Such regions of SEQID NO:2 include, for example, residues 39-44, 252-257, 102-107, 264-269,and 339-344. Exemplary longer peptide immunogens include peptidescomprising residues (i) 131-148, (ii) 230-253, or (iii) 333-355 of SEQID NO:2. Peptide (ii) can be prepared with an additional C-terminal Cysresidue and peptide (iii) with an additional N-terminal Cys residue tofacilitate coupling.

[0051] The immunogenicity of a polypeptide immunogen may be increasedthrough the use of an adjuvant, such as alum (aluminum hydroxide) orFreund's complete or incomplete adjuvant. Polypeptides useful forimmunization also include fusion polypeptides, such as fusions of azvegf4 polypeptide or a portion thereof with an immunoglobulinpolypeptide or with maltose binding protein. If the polypeptide portionis “hapten-like”, such portion may be advantageously joined or linked toa macromolecular carrier (such as keyhole limpet hemocyanin (KLH),bovine serum albumin (BSA), or tetanus toxoid) for immunization.

[0052] Alternative techniques for generating or selecting antibodiesinclude in vitro exposure of lymphocytes to a polypeptide immunogen, andselection of antibody display libraries in phage or similar vectors (forinstance, through use of immobilized or labeled polypeptide). Techniquesfor creating and screening such random peptide display libraries areknown in the art (e.g., Ladner et al., U.S. Pat. No. 5,223,409; Ladneret al., U.S. Pat. No. 4,946,778; Ladner et al., U.S. Pat. No. 5,403,484and Ladner et al., U.S. Pat. No. 5,571,698), and random peptide displaylibraries and kits for screening such libraries are availablecommercially, for instance from Clontech Laboratories (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using the zvegf4sequences disclosed herein to identify proteins that bind to zvegf4.

[0053] Antibodies are determined to be specifically binding if they bindto their intended target (e.g., zvegf4 protein or receptor) with anaffinity at least 10-fold greater than the binding affinity to control(e.g., non-zvegf4 or non-receptor) polypeptide or protein. In thisregard, a “non-zvegf4 polypeptide” includes the related molecules VEGF,VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-A, and PDGF-B, but excludes zvegf4polypeptides from non-human species. Due to the high level of amino acidsequence identity expected between zvegf4 orthologs, antibodies specificfor human zvegf4 may also bind to zvegf4 from other species. The bindingaffinity of an antibody can be readily determined by one of ordinaryskill in the art, for example, by Scatchard analysis (Scatchard, G.,Ann. NY Acad. Sci. 51: 660-672, 1949). Methods for screening andisolating specific antibodies are well known in the art. See, forexample, Paul (ed.), Fundamental Immunology, Raven Press, 1993; Getzoffet al., Adv. in Immunol. 43:1-98, 1988; Goding (ed.), MonoclonalAntibodies: Principles and Practice, Academic Press Ltd., 1996; andBenjamin et al., Ann. Rev. Immunol. 2:67-101, 1984.

[0054] A variety of assays known to those skilled in the art can beutilized to detect antibodies that specifically bind to zvegf4 proteinsor receptors. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassays, inhibition or competition assays, and sandwich assays.

[0055] For therapeutic applications it is generally preferred to useneutralizing antibodies. As used herein, the term “neutralizingantibody” denotes an antibody that inhibits at least 50% of thebiological activity of the cognate antigen when the antibody is added ata 1000-fold molar access. Those of skill in the art will recognize thatgreater neutralizing activity is sometimes desirable, and antibodiesthat provide 50% inhibition at a 100-fold or 10-fold molar access may beadvantageously employed.

[0056] Zvegf4 antagonists also include soluble receptors. As usedherein, a “soluble zvegf4 receptor” is a ligand-binding zvegf4 receptorpolypeptide that is not bound to a cell membrane. Soluble receptors aremost commonly receptor polypeptides that comprise at least a portion ofthe extracellular, ligand binding domain sufficient to bind ligand butlack transmembrane and cytoplasmic domains. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis. Receptor polypeptides are said to be substantially free oftransmembrane and intracellular polypeptide segments when they lacksufficient portions of these segments to provide membrane anchoring orsignal transduction, respectively. Soluble receptors can compriseadditional amino acid residues, such as affinity tags that provide forpurification of the polypeptide or provide sites for attachment of thepolypeptide to a substrate, or immunoglobulin constant region sequences.Dimeric and higher order multimeric soluble receptors are preferred fortheir ability to bind ligand with high affinity. A soluble receptor canbe prepared as a fusion to a dimerizing protein as disclosed in U.S.Pat. Nos. 5,155,027 and 5,567,584. Dimerizing proteins in this regardinclude, for example, immunoglobulin fragments comprising constantregion and hinge domains (e.g., IgG Fc fragments).

[0057] Zvegf4 antagonists further include antisense polynucleotides,which can be used to inhibit zvegf4 gene transcription and therebyinhibit cell activation and/or proliferation in vivo. Polynucleotidesthat are complementary to a segment of a zvegf4-encoding polynucleotide(e.g., a polynucleotide as set forth in SEQ ID NO:1) are designed tobind to zvegf4-encoding mRNA and to inhibit translation of such mRNA.Antisense polynucleotides can be targetted to specific tissues using agene therapy approach with specific vectors and/or promoters, such asviral delivery systems as disclosed in more detail below.

[0058] Ribozymes can also be used as zvegf4 antagonists within thepresent invention. Ribozymes are RNA molecules that contains a catalyticcenter and a target RNA binding portion. The term includes RNA enzymes,self-splicing RNAs, self-cleaving RNAs, and nucleic acid molecules thatperform these catalytic functions. A ribozyme selectively binds to atarget RNA molecule through complementary base pairing, bringing thecatalytic center into close proximity with the target sequence. Theribozyme then cleaves the target RNA and is released, after which it isable to bind and cleave additional molecules. A nucleic acid moleculethat encodes a ribozyme is termed a “ribozyme gene.” Ribozymes can bedesigned to express endonuclease activity that is directed to a certaintarget sequence in a mRNA molecule (see, for example, Draper andMacejak, U.S. Pat. No. 5,496,698, McSwiggen, U.S. Pat. No. 5,525,468,Chowrira and McSwiggen, U.S. Pat. No. 5,631,359, and Robertson andGoldberg, U.S. Pat. No. 5,225,337). An expression vector can beconstructed in which a regulatory element is operably linked to anucleotide sequence that encodes a ribozyme.

[0059] In another approach, expression vectors can be constructed inwhich a regulatory element directs the production of RNA transcriptscapable of promoting RNase P-mediated cleavage of mRNA molecules thatencode a zvegf4 polypeptide. According to this approach, an externalguide sequence can be constructed for directing the endogenous ribozyme,RNase P, to a particular species of intracellular mRNA, which issubsequently cleaved by the cellular ribozyme (see, for example, Altmanet al., U.S. Pat. No. 5,168,053; Yuan et al., Science 263:1269, 1994;Pace et al., WIPO Publication No. WO 96/18733; George et al., WIPOPublication No. WO 96/21731; and Werner et al., WIPO Publication No. WO97/33991). An external guide sequence generally comprises a ten- tofifteen-nucleotide sequence complementary to zvegf4 mRNA, and a 3′-NCCAnucleotide sequence, wherein N is preferably a purine. The externalguide sequence transcripts bind to the targeted mRNA species by theformation of base pairs between the mRNA and the complementary externalguide sequences, thus promoting cleavage of mRNA by RNase P at thenucleotide located at the 5′-side of the base-paired region.

[0060] The growth factor domain of zvegf4 has been found to be an activespecies of the molecule that binds to cell-surface PDGF receptorscompetitively with other PDGF isoforms. Proteolytic processing to removethe N-terminal portion of the molecule is required for this activity.Thus, inhibitors of this proteolytic activation can also be used aszvegf4 antagonists within the present invention.

[0061] For pharmaceutical use, zvegf4 antagonists are formulated fortopical or parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. In general, pharmaceuticalformulations will include a zvegf4 antagonist in combination with apharmaceutically acceptable vehicle, such as saline, buffered saline, 5%dextrose in water, or the like. Formulations may further include one ormore excipients, preservatives, solubilizers, buffering agents, albuminto prevent protein loss on vial surfaces, etc. Methods of formulationare well known in the art and are disclosed, for example, in Remington:The Science and Practice of Phannacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. A “therapeutically effective amount” of acomposition is that amount that produces a statistically significanteffect, such as a statistically significant reduction in diseaseprogression or a statistically significant improvement in organfunction. The exact dose will be determined by the clinician accordingto accepted standards, taking into account the nature and severity ofthe condition to be treated, patient traits, etc. Determination of doseis within the level of ordinary skill in the art. The therapeuticformulations will generally be administered over the period required toachieve a beneficial effect, commonly up to several months and, intreatment of chronic conditions, for a year or more. Dosing is daily orintermittently over the period of treatment. Intravenous administrationwill be by bolus injection or infusion over a typical period of one toseveral hours. Sustained release formulations can also be employed. Fortreatment of pulmonary fibrosis, a zvegf4 antagonist can be delivered byaerosolization according to methods known in the art. See, for example,Wang et al., U.S. Pat. No. 5,011,678; Gonda et al., U.S. Pat. No.5,743,250; and Lloyd et al., U.S. Pat. No. 5,960,792.

[0062] Other mitogenic factors, including the PDGFs, EGF, TGF-β1 andTGF-β2, and FGFs, have been implicated in the initiation or perpetuationof fibrosis. It may therefore be advantageous to combine a zvegf4antagonist with one or more antagonists of these other factors.

[0063] Antibodies are preferably administered parenterally, such as bybolus injection or infusion (intravenous, intramuscular,intraperitoneal, or subcutaneous) over the course of treatment.Antibodies are generally administered in an amount suficient to providea minimum circulating level of antibody throughout the treatment periodof between approximately 20 μg and 1 mg/kg body weight. In this regard,it is preferred to use antibodies having a circulating half-life of atleast 12 hours, preferably at least 4 days, more preferably up to 14-21days. Chimeric and humanized antibodies are expected to have circulatoryhalf-lives of up to four and up to 14-21 days, respectively. In manycases it will be preferable to administer daily doses during a hospitalstay, followed by less frequent bolus injections during a period ofoutpatient treatment. Antibodies can also be delivered by slow-releasedelivery systems, pumps, and other known delivery systems for continuousinfusion. Dosing regimens may be varied to provide the desiredcirculating levels of a particular antibody based on itspharmacokinetics. Thus, doses will be calculated so that the desiredcirculating level of therapeutic agent is maintained. Daily dosesreferred to above may be administered as larger, less frequent bolusadministrations to provide the recited dose averaged over the term ofadministration.

[0064] Those skilled in the art will recognize that the same principleswill guide the use of other zvegf4 antagonists. The dosing regimen for agiven antagonist will be determined by a number of factors includingpotency, pharmacokinetics, and the physicochemical nature of theantagonist. For example, non-peptidic zvegf4 antagonists may beadministered enterally.

[0065] Therapeutic polynucleotides, such as antisense polynucleotides,can be delivered to patients or test animals by way of viral deliverysystems. Exemplary viruses for this purpose include adenovirus,herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus(AAV). Adenovirus, a double-stranded DNA virus, is currently the beststudied gene transfer vector for delivery of heterologous nucleic acids.For review, see Becker et al., Meth. Cell Biol. 43:161-189, 1994; andDouglas and Curiel, Science & Medicine 4:44-53, 1997. The adenovirussystem offers several advantages. Adenovirus can (i) accommodaterelatively large DNA inserts; (ii) be grown to high-titer; (iii) infecta broad range of mammalian cell types; and (iv) be used with manydifferent promoters, including ubiquitous, tissue specific, andregulatable promoters. Because adenoviruses are stable in thebloodstream, they can be administered by intravenous injection.

[0066] By deleting portions of the adenovirus genome, larger inserts (upto 7 kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. When intravenouslyadministered to intact animals, adenovirus primarily targets the liver.If the adenoviral delivery system has an El gene deletion, the viruscannot replicate in the host cells. However, the host's tissue (e.g.,liver) will express and process (and, if a signal sequence is present,secrete) the heterologous protein.

[0067] An alternative method of gene delivery comprises removing cellsfrom the body and introducing a vector into the cells as a naked DNAplasmid. The transformed cells are then re-implanted in the body. NakedDNA vectors are introduced into host cells by methods known in the art,including transfection, electroporation, microinjection, transduction,cell fusion, DEAE dextran, calcium phosphate precipitation, use of agene gun, or use of a DNA vector transporter. See, Wu et al., J. Biol.Chem. 263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963-967,1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.

[0068] Zvegf4 antagonists can be analyzed for receptor-binding activityor inhibition of zvegf4-receptor binding by a variety of methods thatare well known in the art, including receptor competition assays(Bowen-Pope and Ross, Methods Enzymol. 109:69-100, 1985) and through theuse of soluble receptors, including receptors produced as IgG fusionproteins (U.S. Pat. No. 5,750,375). Receptor-binding assays can beperformed on cell lines that contain cell-surface receptors for zvegf4.The receptors can be naturally present in the cell, or can berecombinant receptors expressed by genetically engineered cells.

[0069] Activity of zvegf4 antagonists can be measured in vitro usingcultured cells in assays designed to measure zvegf4 activity.Antagonists will reduce the effects of zvegf4 within the assay.Mitogenic activity can be measured using known assays, including³H-thymidine incorporation assays (as disclosed by, e.g., Raines andRoss, Methods Enzymol. 109:749-773, 1985 and Wahl et al., Mol. CellBiol. 8:5016-5025, 1988), dye incorporation assays (as disclosed by, forexample, Mosman, J. Immunol. Meth. 65:55-63, 1983 and Raz et al., ActaTrop. 68:139-147, 1997) or cell counts. Exemplary mitogenesis assaysmeasure incorporation of ³H-thymidine into (1) 20% confluent cultures tolook for the ability of zvegf4 proteins to further stimulateproliferating cells, and (2) quiescent cells held at confluence for 48hours to look for the ability of zvegf4 proteins to overcomecontact-induced growth inhibition. Exemplary dye incorporation assaysinclude measurement of the incorporation of the dye Alamar blue (Raz etal., ibid.) into target cells. See also, Gospodarowicz et al., J. Cell.Biol. 70:395-405, 1976; Ewton and Florini, Endocrinol. 106:577-583,1980; and Gospodarowicz et al., Proc. Natl. Acad. Sci. USA 86:7311-7315,1989.

[0070] The biological activities of zvegf4 antagonists can be studied innon-human animals by administration of exogenous compounds, byexpression of zvegf4 inhibitory polynucleotides, and by suppression ofendogenous zvegf4 expression through knock-out techniques. Viraldelivery systems (disclosed above) can be employed. Zvegf4 antagonistscan be administered or expressed individually, in combination with otherzvegf4 antagonists, or in combination other compounds, including othergrowth factor antagonists. Test animals are monitored for changes insuch parameters as clinical signs, body weight, blood cell counts,clinical chemistry, histopathology, and the like.

[0071] Effects of zvegf4 antagonists on liver and kidney fibrosis can betested in known animal models, such as the db/db mouse model disclosedby Cohen et al., Diabetologia 39:270-274, 1996 and Cohen et al., J.Clin. Invest. 95:2338-2345, 1995, or transgenic animal models (Imai etal., Contrib. Nephrol. 107:205-215, 1994).

[0072] Effects on lung fibrosis can also be assayed in a mouse modelusing bleomycin. The chemotherapy agent bleomycin is a known causativeagent of pulmonary fibrosis in humans and can induce interstitial lungdisease in mice, including an increase in the number of fibroblasts,enhanced collagen deposition, and dysregulated matrix remodeling.C57B1/6 mice are administered bleomycin by osmotic minipump for 1 week.There follows a period of inflammation, with cutaneous toxicitybeginning approximately 4-7 days after bleomycin administration andcontinuing for about a week, after which the mice appear to regainhealth. About 3-4 weeks after the finish of bleomycin delivery, the miceare sacrificed, and the lungs are examined histologically for signs offibrosis. Scoring is based on the extent of lung fibrotic lesions andtheir severity. Serum is assayed for lactic dehydrogenase, anintracellular enzyme that is released into the circulation upon generalcell death or injury. Lung tissue is assayed for hydroxyproline as ameasure of collagen deposition.

[0073] Mice and other animals carrying a zvegf4-expressing adenovirusvector are also useful models for testing zvegf4 antagonists and otherantifibroproliferative agents.

[0074] The invention is further illustrated by the following,non-limiting examples.

EXAMPLES Example 1

[0075] Zvegf4 was identified from the sequence of a clone from a humanchronic myelogenous leukemia cell (K562) library by its homology to theVEGF family. Additional sequence was elucidated from a long sequenceread of a clone from a pituitary library. An antisense expressedsequence tag (EST) for zvegf4 was found, for which its 5′ partner wasidentified. This 5′ EST (EST448186; GenBank) appeared to contain the 5′untranslated sequence for zvegf4. A primer was designed from EST448186to close the gap in the sequence. 20 μm each of oligonucleotidesZC21,987 (SEQ ID NO:5) and ZC21,120 (SEQ ID NO:6), and 1.93 μg of athyroid library were used in the PCR reaction with 5% DMSO and {fraction(1/10)} volume of a commercial reagent (GC-Melt™; Clontech Laboratories,Inc., Palo Alto, Calif.). The reaction was run for 1 minute at 94degrees; then 30 cycles of 94 degrees, 20 seconds; 67 degrees, 1 minute;then a final 5-minute incubation at 72 degrees. A resulting 833-bpproduct was sequenced and found to be a zvegf4 fragment containing theremainder of the coding sequence with an intiation MET codon, upstreamstop codon, and 5′ untranslated sequence. The composite sequenceincluded an open reading frame of 1,110 bp (SEQ ID NO:1).

Example 2

[0076] A partial mouse zvegf4 sequence was obtained by probing a mousegenomic library (obtained from Clontech Laboratories, Inc.) with a 1,289bp EcoRI human zvegf4 restriction digest fragment containing the entirecoding sequence. The probe was labeled with ³²P using a commerciallyavailable kit (Rediprime™ II random-prime labeling system; AmershamPharmacia, Buckinghamshire, England). Unincorporated radioactivity wasremoved using a commercially available push column (NucTrap® column;Stratagene, La Jolla, Calif.; see U.S. Pat. No. 5,336,412). Twenty-fourfilter lifts were prehybridized overnight at 50° C. in a hybridizationsolution (ExpressHyb™ Hybridization Solution; Clontech Laboratories,Inc.) containing 0.1 mg/ml salmon sperm DNA that had been boiled 5minutes, then iced. Filters were hybridized overnight at 50° C. inhybridization solution (ExpressHyb™) containing 1.0×10⁶ cpm/ml zvegf4probe, 0.1 mg/ml salmon sperm DNA, and 0.5 μg/ml mouse cot-1 DNA thathad been boiled 5 minutes, then iced. Filter lifts were washed in 2×SSC,0.1% SDS at room temperature for 2 hours, then the temperature wasraised to 60° C. for one hour. Overnight exposure at −80° C. showed 7putative primary hits.

[0077] Four of the primary hits were plated on a lawn of E. coli K802cells (obtained from Clontech Laboratories, Inc.). Filter lifts wereprepared and hybridized overnight with the human zvegf4 probe. Two ofthe 4 primary putative hits that were tested came up positive.

[0078] DNA was prepared from one positive plaque and digested with BamHIand PstI. The digest was run on a 1% Tris-Borate-EDTA gel, and a 2.0 kbdoublet and 2.7 kb/2.9 kb bands were excised from the gel and extractedfrom the agarose by conventional methods. Both 2.0 kb fragments werefound to strongly hybridize to the human zvegf4 probe. These fragmentswere sequenced and found to contain part of the mouse zvegf4 CUB domain.Primers were designed from the sequence for use in a PCR cDNA screen.

[0079] A panel of mouse cDNAs was screened by PCR with primers ZC26,317(SEQ ID NO:7) and ZC26,318 (SEQ ID NO:8). Embryo, salivary gland,neonatal skin, and testis showed strong products of the predicted 200 bpsize.

[0080] Mouse testis and salivary gland libraries were screened by PCRusing primers ZC26,317 (SEQ ID NO:7) and ZC26,318 (SEQ ID NO:8). Thetestis library yielded one clone, named “zvegf4mpzp7x-6”, that wasincomplete at the 5′ end and appeared to contain an intron at the 5′end. The salivary gland library yielded one clone, named“zvegf4mpzp7x-7”, that had a 225-bp deletion in coding compared to clonezvegf4mpzp7x-6. The sequences derived from zvegf4mpzp7x-6 andzvegf4mpzp7x-7 were combined to produce a full-length mouse zvegf4polynucleotide sequence (SEQ ID NO:3) and mouse zvegf4 polypeptidesequence (SEQ ID NO:4).

[0081] A full-length cDNA clone was generated by a two-step ligation offragments from the two clones. An EcoRI/HindIII 3′ fragment was preparedfrom clone zvegf4mpzp7x-6. The 528-bp fragment was gel-purified andligated into a phagemid vector (pBluescript® II KS(+); Stratagene) thathad been digested with EcoRI and HindIII. Three μg of the resultingconstruct was digested with 15 units of EcoRI. The linearized plasmidwas purified and ligated with a 754-bp 5′ EcoRI fragment from clonezvegf4mpzp7x-7.

Example 3

[0082] Recombinant human zvegf4 having a carboxyl-terminal Glu-Gluaffinity tag was produced in a baculovirus expression system accordingto conventional methods. The culture was harvested, and the cells werelysed with a solution of 0.02 M Tris-HCl, pH 8.3, 1 mM EDTA, 1 mM DTT, 1mM 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride (Pefabloc®SC; Boehringer-Mannheim), 0.5 μM aprotinin, 4 mM leupeptin, 4 mM E-64,1% NP-40 at 4° C. for 15 minutes on a rotator. The solution wascentrifuged, and the supernatant was recovered. Twenty ml of extract wascombined with 50 μl of anti-Glu-Glu antibody conjugated to derivatizedagarose beads (Sepharose®; Amersham Pharmacia Biotech Inc., Piscataway,N.J.) in 50 μl buffer. The mixture was incubated on a rotator at 4° C.overnight. The beads were recovered by centrifugation and washed 3×15minutes at 4° C. Pellets were combined with sample buffer containingreducing agent and heated at 98° C. for five minutes. The protein wasanalyzed by polyacrylamide gel electrophoresis under reducing conditionsfollowed by western blotting on a PVDF membrane using an antibody to theaffinity tag. Two bands were detected, one a M_(r)≈49 kD and the otherat M_(r)≈21 kD. Sequence analysis showed the larger band to comprise twosequences, one beginning at Arg-19 of SEQ ID NO:2 and the otherbeginning at Asn-35 of SEQ ID NO:2. The asparagine residue appeared tohave been deamidated to an aspartic acid. The smaller band began atSer-250 of SEQ ID NO:2.

Example 4

[0083] To prepare adenovirus vectors, the protein coding region ofzvegf4 is amplified by PCR using primers that add FseI and AscIrestriction sites at the 5′ and 3′ termini, respectively. PCR primersare used with a template containing the full-length zvegf4 cDNA in a PCRreaction as follows: incubation at 95° C. for 5 minutes; followed by 15cycles at 95° C. for 1 min., 58° C. for 1 min., and 72° C. for 1.5 min.;followed by 72° C. for 7 min.; followed by a 4° C. soak. The reactionproducts are loaded onto a 1.2% low-melt agarose (SeaPlaque GTG™; FMC,Rockland, Me.) gel in TAE buffer. The zvegf4 PCR product is excised fromthe gel and purified using a spin column containing a silica gelmembrane (QIAquick™ Gel Extraction Kit; Qiagen, Inc., Valencia, Calif.)as per kit instructions. The zvegf4 product is then digested,phenol/chloroform extracted, EtOH precipitated, and rehydrated in 20 mlTE (Tris/EDTA pH 8). The zvegf4 fragment is then ligated into thecloning sites of the transgenic vector pTG12-8. Vector pTG12-8 wasderived from p2999B4 (Palmiter et al., Mol. Cell Biol. 13:5266-5275,1993) by insertion of a rat insulin II intron (ca. 200 bp) andpolylinker (Fse I/Pme I/Asc I) into the Nru I site. The vector comprisesa mouse metallothionein (MT-1) promoter (ca. 750 bp) and human growthhormone (hGH) untranslated region and polyadenylation signal (ca. 650bp) flanked by 10 kb of MT-1 5′ flanking sequence and 7 kb of MT-1 3′flanking sequence. The construct is transformed into E. coli host cells(Electromax DH10BTM cells; obtained from Life Technologies, Inc.,Gaithersburg, Md.) by electroporation. Clones containing zvegf4 DNA areidentified by restriction analysis. A positive clone is confirmed bydirect sequencing.

[0084] The zvegf4 cDNA is released from the pTG12-8 vector using FseIand AscI enzymes. The cDNA is isolated on a 1% low melt agarose gel, andis then excised from the gel. The gel slice is melted at 70° C.,extracted twice with an equal volume of Tris-buffered phenol, and EtOHprecipitated. The DNA is resuspended in 10 μl H₂O.

[0085] The zvegf4 cDNA is cloned into the FseI-AscI sites of a modifiedpAdTrack CMV (He et al., Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998).This construct contains a green fluorescent protein (GFP) marker gene.The CMV promoter driving GFP expression has been replaced with the SV40promoter, and the SV40 polyadenylation signal has been replaced with thehuman growth hormone polyadenylation signal. In addition, the nativepolylinker has been replaced with FseI, EcoRV, and AscI sites. Thismodified form of pAdTrack CMV is named pZyTrack. Ligation is performedusing a DNA ligation and screening kit (Fast-Link™; EpicentreTechnologies, Madison, Wis.). In order to linearize the plasmid,approximately 5 μg of the pZyTrack zvegf4 plasmid is digested with PmeI.Approximately 1 μg of the linearized plasmid is cotransformed with 200ng of supercoiled pAdEasy (He et al., ibid.) into BJ5183 cells. Theco-transformation is done using a Bio-Rad Gene Pulser at 2.5 kV, 200ohms and 25 μF. The entire co-transformation is plated on 4 LB platescontaining 25 μg/ml kanamycin. The smallest colonies are picked andexpanded in LB/kanamycin, and recombinant adenovirus DNA is identifiedby standard DNA miniprep procedures. Digestion of the recombinantadenovirus DNA with FseI and AscI confirms the presence of zvegf4 DNA.The recombinant adenovirus miniprep DNA is transformed into E. coliDH10B competent cells, and DNA is prepared therefrom.

[0086] Approximately 5 μg of recombinant adenoviral DNA is digested withPacI enzyme (New England Biolabs) for 3 hours at 37° C. in a reactionvolume of 100 μl containing 20-30U of PacI. The digested DNA isextracted twice with an equal volume of phenol/chloroform andprecipitated with ethanol. The DNA pellet is resuspended in 10 μldistilled water. A T25 flask of QBI-293A cells (Quantum Biotechnologies,Inc., Montreal, Canada), inoculated the day before and grown to 60-70%confluence, are transfected with the PacI digested DNA. ThePacI-digested DNA is diluted up to a total volume of 50 μl with sterileHBS (150 mM NaCl, 20 mM HEPES). In a separate tube, 20 μl of 1 mg/mlN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate(DOTAP; Boehringer Mannheim) is diluted to a total volume of 100 μl withHBS. The DNA is added to the DOTAP, mixed gently by pipeting up anddown, and left at room temperature for 15 minutes. The media is removedfrom the 293A cells and washed with 5 ml serum-free MEM-alpha (LifeTechnologies, Gaithersburg, Md.) containing 1 mM sodium pyruvate (LifeTechnologies), 0.1 mM MEM non-essential amino acids (Life Technologies)and 25 mM HEPES buffer (Life Technologies). 5 ml of serum-free MEM isadded, and the cells are held at 37° C. The DNA/lipid mixture is addeddrop-wise to the flask, mixed gently, and incubated at 37° C. for 4hours. After 4 hours the media containing the DNA/lipid mixture isaspirated off and replaced with 5 ml complete MEM containing 5% fetalbovine serum. The transfected cells are monitored for GFP expression andinformation of foci (viral plaques).

[0087] Seven days after transfection of 293A cells with the recombinantadenoviral DNA, cells expressing GFP start to form foci. The crude virallysate is harvested by using a cell scraper to collect the cells. Thelysate is transferred to a 50 ml conical tube. To release most of thevirus particles from the cells, three freeze/thaw cycles are done in adry ice/ethanol bath and a 37° C. waterbath.

[0088] Ten 10-cm plates of nearly confluent (80-90%) 293A cells are setup 20 hours prior to infection. The crude lysate is amplified (primaryamplification) to obtain a working stock of zvegf4 recombinantadenovirus (rAdV) lysate. 200 ml of crude rAdV lysate is added to each10-cm plate, and the plates are monitored for 48 to 72 hours looking forcytopathic effect (CPE) under the white light microscope and expressionof GFP under the fluorescent microscope. When all of the cells show CPE,this 1° stock lysate is collected, and freeze/thaw cycles are performedas described above.

[0089] Secondary (2°) amplification of zvegf4 rAdV is obtained fromtwenty 15-cm tissue culture dishes of 80-90% confluent 293A cells. Allbut 20 ml of 5% MEM media is removed, and each dish is inoculated with300-500 ml of 1° amplified rAdv lysate. After 48 hours the cells arelysed from virus production, the lysate is collected into 250 mlpolypropylene centrifuge bottles, and the rAdV is purified.

[0090] NP-40 detergent is added to a final concentration of 0.5% to thebottles of crude lysate to lyse all cells. Bottles are placed on arotating platform for 10 minutes and agitated as fast as possible. Thedebris is pelleted by centrifugation at 20,000×G for 15 minutes. Thesupernatant is transferred to 250-ml polycarbonate centrifuge bottles,and 0.5 volume of 20% PEG8000/2.5M NaCl solution is added. The bottlesare shaken overnight on ice. The bottles are centrifuged at 20,000×G for15 minutes, and the supernatants are discarded into a bleach solution. Awhite precipitate (precipitated virus/PEG) forms in two vertical linesalong the walls of the bottles on either side of the spin mark. Using asterile cell scraper, the precipitate from 2 bottles is resuspended in2.5 ml PBS. The virus solution is placed in 2-ml microcentrifuge tubesand centrifuged at 14,000×G in a microcentrifuge for 10 minutes toremove any additional cell debris. The supernatants from the 2-mlmicrocentrifuge tubes are transferred into a 15-ml polypropylene snapcaptube and adjusted to a density of 1.34 g/ml with CsCl. The volume of thevirus solution is estimated, and 0.55 g/ml of CsCl is added. The CsCl isdissolved, and 1 ml of this solution is weighed. The solution istransferred to polycarbonate, thick-walled, 3.2 ml centrifuge tubes(Beckman) and spun at 348,000×G for 3-4 hours at 25° C. The virus formsa white band. Using wide-bore pipette tips, the virus band is collected.

[0091] The virus recovered from the gradient includes a large amount ofCsCl, which must be removed before it can be used on cells. PharmaciaPD-10 columns prepacked with Sephadex® G-25M (Pharmacia) are used todesalt the virus preparation. The column is equilibrated with 20 ml ofPBS. The virus is loaded and allowed to run into the column. 5 ml of PBSis added to the column, and fractions of 8-10 drops collected. Theoptical density of a 1:50 dilution of each fraction is determined at 260nm on a spectrophotometer, and a clear absorbance peak is identified.Peak fractions are pooled, and the optical density (OD) of a 1:25dilution is determined. OD is converted into virus concentration usingthe formula (OD at 260 nm)(25)(1.1×10¹²)=virions/ml.

[0092] To store the virus, glycerol is added to the purified virus to afinal concentration of 15%, mixed gently and stored in aliquots at −80°C.

[0093] A protocol developed by Quantum Biotechnologies, Inc. (Montreal,Canada) is followed to measure recombinant virus infectivity. Briefly,two 96-well tissue culture plates are seeded with 1×10⁴ 293A cells perwell in MEM containing 2% fetal bovine serum for each recombinant virusto be assayed. After 24 hours, 10-fold dilutions of each virus from1×10⁻² to 1×10⁻¹⁴ are made in MEM containing 2% fetal bovine serum. 100μl of each dilution is placed in each of 20 wells. After 5 days at 37°C., wells are read either positive or negative for CPE, and PFU/ml iscalculated.

[0094] TCID₅₀ formulation used is as per Quantum Biotechnologies, Inc.,above. The titer (T) is determined from a plate where virus used isdiluted from 10⁻² to 10⁻¹⁴, and read 5 days after the infection. At eachdilution a ratio (R) of positive wells for CPE per the total number ofwells is determined. The titer of the undiluted sample isT=10^((1+F))=TCID₅₀/ml, where F=1+d(S−0.5), S is the sum of the ratios(R), and d is Log₁₀ of the dilution series (e.g., d=1 for a ten-folddilution series). To convert TCID₅₀/ml to pfu/ml, 0.7 is subtracted fromthe exponent in the calculation for titer (T).

Example 5

[0095] The human zvegf4 cDNA was cloned into the EcoRV-AscI sites ofpZyTrack (Example 4). Ligation was performed using a commerciallyavailable DNA ligation and screening kit (Fast-Link™ kit; EpicentreTechnologies, Madison, Wis.).

[0096] Zvegf4 was assayed in an aortic ring outgrowth assay (Nicosia andOttinetti, Laboratory Investigation 63:115, 1990; Villaschi and Nicosia,Am. J. Pathology 143:181-190, 1993). Thoracic aortas were isolated from1-2 month old Sprague-Dawley male rats and transferred to petri dishescontaining HANK's buffered salt solution. The aortas were flushed withadditional HANK's buffered salt solution to remove blood, andadventitial tissue surrounding the aortas was carefully removed. Cleanedaortas were transferred to petri dishes containing EBM basal media,serum free (Clonetics, San Diego, Calif.). Aortic rings were obtained byslicing approximately 1-mm sections using a scalpel blade. The ends ofthe aortas used to hold them in place were not used. The rings wererinsed in fresh EBM basal media and placed individually in wells of a24-well plate coated with basement membrane matrix (Matrigel®; BectonDickinson, Franklin Lakes, N.J.). The rings were overlayed with anadditional 50 μl of the matrix solution and placed at 37° C. for 30minutes to allow the matrix to gel. Test samples were diluted in EBMbasal serum-free media supplemented with 100 units/ml penicillin, 100μg/ml streptomycin and HEPES buffer and added at 1 ml/well. Backgroundcontrol was EBM basal serum-free media alone. Basic FGF (R&D Systems,Minneapolis, Minn.) at 20 ng/ml was used as a positive control. Zvegf4adenovirus was added to wells, assuming a cell count of 500,000 cellsand a multiplicity of infection of 5000 particles/cell. A nulladenovirus (designated “zPar”) was used as a control. Samples were addedin a minimum of quadruplets. Rings were incubated for 5-7 days at 37° C.and analyzed for growth. Aortic outgrowth was scored by multiple,blinded observers using 0 as no growth and 4 as maximum growth. Zvegf4adenovirus produced a significant increase in outgrowth, comparable tothe bFGF control.

[0097] Zvegf4 adenovirus infection produced a significant increase inthe outgrowth of cells as compared to parental virus control. Cellsisolated from the matrix surrounding the aortic ring were identified asfibroblasts or smooth muscle cells (SMC) by staining for alpha smoothmuscle actin (characteristic of SMCs), and vimentin and type I collagen(characteristic of fibroblasts). In contrast, there were no endothelialcells detected as indicated by the absence of staining for vonWillebrand's factor, a specific endothelial marker.

[0098] Potent induction of cellular outgrowth, similar to that inducedby purified PDGF-AA and PDGF-BB, was also observed following treatmentwith purified growth factor domain (mature) zvegf4. These patterns ofoutgrowth were unlike that seen following VEGF treatment, which producedsparser endothelial sprouts. The ability of zvegf4 to induce a responsesimilar to that of PDGF-AA and PDGF-BB, that is a smooth muscle andfibroblast migratory and cyto-kinetic response, correlated with theinvolvement of PDGF receptor stimulation in fibroproliferative responsesof the vasculature.

Example 6

[0099] Polyclonal anti-peptide antibodies were prepared by immunizing 2female New Zealand white rabbits with the peptides huzvegf4-1 (SEQ IDNO:9), huzvegf4-2 (SEQ ID NO:10), or huzvegf4-3 (SEQ ID NO:11). Thepeptides were synthesized using an Applied Biosystems Model 431A peptidesynthesizer (Applied Biosystems, Inc., Foster City, Calif.) according tothe manufacturer's instructions. The peptides were conjugated to keyholelimpet hemocyanin (KLH) with maleimide activation. The rabbits were eachgiven an initial intraperitoneal (ip) injection of 200 μg of peptide inComplete Freund's Adjuvant followed by booster ip injections of 100 μgpeptide in Incomplete Freund's Adjuvant every three weeks. Seven to tendays after the administration of the second booster injection (3 totalinjections), the animals were bled, and the sera were collected. Theanimals were then boosted and bled every three weeks.

[0100] The zvegf4 peptide-specific rabbit sera were characterized by anELISA titer check using 1 μg/ml of the peptide used to make the antibodyas an antibody target. The 2 rabbit sera to the huzvegf4-1 peptide hadtiter to their specific peptide at a dilution of 1:5,000,000. The 2rabbit sera to the huzvegf4-2 peptide had titer to their specificpeptide at a dilution of 1:5,000,000. The 2 rabbit sera to thehuzvegf4-3 peptide had titer to their specific peptide at a dilution of1:500,000.

[0101] The zvegf4 peptide-specific polyclonal antibodies were affinitypurified from the sera using CNBr-Sepharose® 4B protein columns(Pharmacia LKB) that were prepared using 10 mg of the specific peptideper gram CNBr-Sepharose®, followed by 20× dialysis in PBS overnight.Zvegf4-specific antibodies were characterized by an ELISA titer checkusing 1 μg/ml of the appropriate peptide antigens as antibody targets.The lower limit of detection (LLD) of the anti-huzvegf4-1 affinitypurified antibody on its specific antigen (huzvegf4-1 peptide) was adilution of 0.1 μg/ml. The LLD of the anti-huzvegf4-2 affinity purifiedantibody on its specific antigen (huzveg4-2 peptide) was a dilution of 5ng/ml. The LLD of the rabbit anti-huzvegf4-3 affinity purified antibodyon its specific antigen (huzvegf4-3 peptide) was a dilution of 5 ng/ml.

Example 7

[0102] Recombinant amino-terminally Glu-Glu-tagged zvegf4 growth factordomain with an amino-terminal Glu-Glu (EYMPME; SEQ ID NO:12) tag(zvegf4-nee-GFD) was produced from recombinant baculovirus-infectedinsect cells. 28-liter cultures were harvested, and the media werefiltered using a 0.45 μm filter. Protein was purified from theconditioned media by a combination of cation-exchange chromatography,antibody affinity chromatography, and size-exclusion chromatography.Cultured medium (pH 7.0, conductivity 9 mS) was directly loaded onto a25-ml cation exchange column (Poros® 50 HS; PerSeptive Biosystems,Framingham, Mass.). The column was washed with ten column volumes (cv)of PBS, and the bound protein was eluted with a gradient of 20-100% of750 mM NaCl in PBS (Buffer B) for 15 cv followed by 5 cv of 100% BufferB at 5 mil/min. Five-ml fractions were collected. Samples from thecolumn were analyzed by SDS-PAGE with silver staining and westernblotting for the presence of zvegf4-nee-GFD. Zvefg4-nee-GFD-containingfractions were pooled and loaded onto an 8-mi anti-Glu-Glu antibodycolumn and eluted with 50 ml of 0.5 mg/ml EYMPTD (SEQ ID NO:13) peptide(obtained from Princeton Biomolecules Corporation, Langhorne, Pa.) inPBS. One-ml fractions were pooled and concentrated to 4 ml using using aBiomax™ −5 concentrator (Millipore Corp., Bedford, Mass.) and loadedonto a 16×1000 mm gel filtration column (Sephacryl™ S-100 HR; AmershamPharmacia Biotech, Piscataway, N.J.) at 1.5 ml/minute. Five-ml fractionscontaining purified zvegf4-nee-GFD were pooled, filtered through a 0.2μm filter, aliquoted into 100 μl aliquots, and frozen at −80° C. Theconcentration of the final purified protein was determined by BCA assay(Pierce Chemical Co., Rockford, Ill.) to be 0.4 mg/ml, and the yield wascalculated to be 8.4 mg.

[0103] Recombinant zvegf4-nee-GFD was analyzed by SDS-PAGE (Nupage™4-12% gel; Novex, San Diego, Calif.) with silver staining (FASTsilver™,Geno Technology, Inc., Maplewood, Mo.) and Western blotting usingantibodies to the peptide tag. Conditioned media or purified protein waselectrophoresed using an electrophoresis mini-cell (XCell II™ mini-cell;Novex) and transferred to nitrocellulose (0.2 μm; Novex) at roomtemperature using a blot module (XCell II™; Novex) with stirringaccording to directions provided in the instrument manual. The transferwas run at 500 mA for one hour in a buffer containing 25 mM Tris base,200 mM glycine, and 20% methanol. The filters were then blocked with 10%non-fat dry milk in PBS for 10 minutes at room temperature. Thenitrocellulose was quickly rinsed, then the mouse anti-peptide primaryantibody was added, diluted 1:1000 in PBS containing 2.5% non-fat drymilk. The blots were incubated for two hours at room temperature orovernight at 4° C. with gentle shaking. Following the incubation, blotswere washed three times for 10 minutes each in PBS, then labeled with asecondary antibody (goat anti-mouse IgG conjugated to horseradishperoxidase) diluted 1:1000 in PBS containing 2.5% non-fat dry milk, andthe blots were incubated for two hours at room temperature with gentleshaking. The blots were then washed three times, 10 minutes each, inPBS, then quickly rinsed with H₂O. The blots were developed usingcommercially available chemiluminescent substrate reagents (SuperSignal®ULTRA reagents 1 and 2 mixed 1:1; reagents obtained from Pierce ChemicalCo.), and the signal was captured using image analysis software(Lumi-Imager™ Lumi Analyst 3.0; Boehringer Mannheim GmbH, Germany) fortimes ranging from 10 seconds to 5 minutes or as necessary.

[0104] The purified zvefg4-nee-GFD appeared as two bands on thesilver-stained gel at about 31 and 17 kDa under non-reducing conditionsand as a single band of 17 kDa under reducing conditions. This suggestedexistence of a dimeric form of zvegf4-nee-GFD under non-reducingconditions. The purified protein consisted of approximately 90% dimerand 10% monomer.

Example 8

[0105] Recombinant human zvegf4 (expressed from the full-length codingsequence) was analyzed for mitogenic activity on rat liver stellatecells (Greenwel et al., Laboratory Invest. 65:644, 1991; Greenwel etal., Laboratory Invest. 69:210, 1993), human aortic smooth muscle cells(Clonetics Corp., Walkersville, Md.), human retinal pericytes (CloneticsCorp.) and human hepatic fibroblasts (Clonetics Corp.). Test samplesconsisted of conditioned media (CM) from adenovirally infected HaCaThuman keratinocyte cells (Boukamp et al., J. Cell. Biol. 106:761-771,1988; Skobe and Fusenig, Proc. Natl. Acad. Sci. USA 95:1050-1055, 1998;obtained from Dr. Norbert E. Fusenig, Deutsches Krebsforschungszentrum,Heidelberg, Germany) expressing full length zvegf4. Control CM wasgenerated from HaCaT cells infected with a parental GFP-expressingadenovirus (zPar). The CM were concentrated 10-fold using a 15-mlcentrifugal filter device with a 10K membrane filter (Ultrafree®;Millipore Corp., Bedford, Mass.), then diluted back to 1× with ITSmedium (serum-free DMEM/Ham's F-12 medium containing 5 μg/ml insulin, 20μg/ml transferrin, and 16 pg/ml selenium). Cells were plated at adensity of 2,000 cells/well in 96-well culture plates and grown forapproximately 72 hours in DMEM containing 10% fetal calf serum at 37° C.Cells were quiesced by incubating them for approximately 20 hours in ITSmedium. At the time of the assay, the medium was removed, and testsamples were added to the wells in triplicate. For measurement of[³H]thymidine incorporation, 20 μl of a 50 μCi/ml stock in DMEM wasadded directly to the cells, for a final activity of 1 μCi/well. Afteranother 24-hour incubation, media were removed and cells were incubatedwith 0.1 ml of trypsin until cells detached. Cells were harvested onto96-well filter plates using a sample harvester (FilterMate™ harvester;Packard Instrument Co., Meriden, Conn.). The plates were then dried at65° C. for 15 minutes, sealed after adding 40 μl/well scintillationcocktail (Microscint™ O; Packard Instrument Co.) and counted on amicroplate scintillation counter (Topcount®; Packard Instrument Co.).Results, presented in Table 2, demonstrated that zvegf4 CM hadapproximately 7-fold higher mitogenic activity than control CM onpericytes cells and approximately a 1.5-2.4-fold higher mitogenicactivity on the other cell types tested. While not wishing to be boundby theory, it is believed that the observed activity may be due to thepresence of cleaved zvegf4 protein (i.e., growth factor domain). TABLE 2CPM incorporated Zvegf4 (1× CM) zPar (1× CM) Sample Mean St. dev. MeanSt. dev. Human retinal pericytes 3621 223 523 306 Human hepaticfibroblasts 7757 753 3232 264 Human aortic SMC 2009 37 1263 51 Rat liverstellate cells 34707 1411 14413 1939

Example 9

[0106] Recombinant, C-terminally glu-glu tagged, human zvegf4 (expressedin baculovirus-infected cells expressing a full-length zvegf4 codingsequence) was analyzed for mitogenic activity on human aortic smoothmuscle cells (HAoSMC) (Clonetics), human retinal pericytes (Clonetics)and human aortic adventitial fibroblasts (AoAF) (Clonetics). Cells wereplated at a density of 2,000 cells/well in 96-well culture plates andgrown for approximately 72 hours in DMEM containing 10% fetal calf serumat 37° C. Cells were quiesced by incubating them for 20 hours in ITSmedium. At the time of the assay, the medium was removed, and testsamples were added to the wells in triplicate. Purified protein in abuffer containing 0.1% BSA was serially diluted into ITS medium atconcentrations of 1 μg/ml to 1 ng/ml and added to the test plate. Acontrol buffer of 0.1% BSA was diluted identically to the highestconcentration of zvegf4 protein and added to the plate. For measurementof [³H]thymidine incorporation, 20 μl of a 50 μCi/ml stock in DMEM wasadded directly to the cells, for a final activity of 1 μCi/well. Afteranother 24-hour incubation, mitogenic activity was assessed by measuringthe uptake of [³H]thymidine. Media were removed, and cells wereincubated with 0.1 ml of trypsin until cells detached. Cells wereharvested onto 96-well filter plates using a sample harvester(FilterMate™ harvester; Packard Instrument Co., Meriden, Conn.). Theplates were then dried at 65° C. for 15 minutes, sealed after adding 40μl/well scintillation cocktail (Microscint™ O; Packard Instrument Co.)and counted on a microplate scintillation counter (Topcount®; PackardInstrument Co.). Results, presented in Table 3, demonstrated that 80ng/ml zvegf4 had approximately 1.7-fold higher mitogenic activity onpericytes, 3.2-fold higher activity on aortic SMCs, and 2.6-fold higheractivity on aortic fibroblasts as compared to the buffer control. TABLE3 CPM Incorporated Pericytes HAoSMC AoAF Sample Mean St. dev. Mean St.dev. Mean St. dev. Zvegf4, 80 96.7 18.2 488.7 29.6 177.0 1.0 ng/mlZvegf4, 20 81.7 11.7 211.7 50.8 107.7 20.1 ng/ml Zvegf4, 5 67.3 6.7191.7 4.5 123.7 10.5 ng/ml Buffer control 58.7 8.5 152.3 40.1 68.7 8.3

Example 10

[0107] The protein-coding region of human zvegf4 DNA was amplified byPCR using primers that added PmeI and AscI restriction sites at the 5′and 3′ termini, respectively. The resulting zvegf4 cDNA was cloned intothe EcoRV-AscI sites of pZyTrack (Example 4). Recombinant adenovirus wasgenerated in 293A cells and purified on CsCl gradients. Viral particlenumbers were determined by spectrophotometry, and infectious particlenumbers were determined by TCID₅₀ assay. The virus was designatedAdZyvegf4.

[0108] Eight-week-old C57BL/6 mice were infected with AdZyvegf4 todetermine the effects on serum chemistry, complete blood counts (CBC),body and organ weight changes, and histology. On day −1, the mice weretagged, individually weighed, and group normalized for separation intotreatment groups (4 mice per cage). Group 1 mice (n=8 females, 7 males)received GFP (control) adenovirus, 1×10¹¹ particles. Group 2 mice (n=8females, 6 males) received zvegf4 adenovirus, 1×10¹¹ particles. Group 3mice (n=8 females, 8 males) were untreated controls. On day 0, the micereceived injections of the appropriate adenovirus solution. On day 10,blood was collected (under ether anesthesia) for CBCs and clinicalchemistry measurements. On day 20, mice were weighed and sacrificed bycervical dislocation after collecting blood (under ether anesthesia) forCBCs and clinical chemistry measurements. Selected tissues were fixedand evaluated for morphological changes. The following pathologicalfindings were noted in the majority (80-100%) of the animals treatedwith the AdZyvegf4 adenovirus, and were not observed in either of theother two groups.

[0109] In the liver, there was moderate proliferation of sinusoidalcells, especially cells with small ovoid nuclei and no observablecytoplasm lining the sinusoids that were more clustered in the venousregions of the hepatic lobule. The cells appeared to be spindle Ito (orstellate) cells, which are a major cell type incriminated in the onsetand progression of hepatic fibrosis.

[0110] In all AdZyvegf4-treated animals, the glomeruli of the kidneyswere enlarged and were characterized by increased cellularity, diagnosedas proliferative glomerulopathy. Because of their location andmorphological characteristics, the proliferating cells within theglomerulus that contributed to its enlargement were most likelymesangial cells. In addition, there was evidence of tubular regenerationin many of the kidneys, characterized by tubular epithelial cells withincreased basophilia.

[0111] An increased amount of bronchoalveolar lymphoid tissue was notedin the lungs of the AdZyvegf4-treated animals. Bronchoalveolar lymphoidtissue consisted predominantly of clusters of lymphocytes admixed withfewer numbers of plasma cells around vessels within the lung parenchyma,a sign of lung inflammatory response, which is an important initiatorand participant in several forms of lung fibrosis.

[0112] In the femur, the majority of animals displayed minimal to severeendosteal bone filling the marrow space, with decreased amounts ofhematopoietic elements resulting from loss of marrow space due to theproliferating endosteal bone. In addition, four of six male and two ofeight female animals had proliferation of stromal cells, which wascharacterized by an increased number of spindle-shaped cells.

Example 11

[0113] 90 μg of full-length, recombinant human zvegf4 protein wasdissolved in 500 μl PBS containing 2 mCi Na¹²⁵I (Amersham Corp.). Onederivatized, nonporous polystyrene bead (IODO-Beads®; Pierce, Rockford,Ill.) was added, and the reaction mixture was incubated one minute onice. The iodinated protein was separated from unincorporated ¹²⁵I by gelfiltration using an elution buffer of 10% acetic acid, 150 mM NaCl, and0.25% gelatin. The active fraction contained 29 μg/ml ¹²⁵I-zvegf4 with aspecific activity of 3.0×10⁴ cpm/ng.

[0114] The following cell lines were plated into the wells of a 24-welltissue culture dish and cultured in growth medium for three days:

[0115] 1. Human retinal pericytes, passage 6 (pericytes).

[0116] 2. Rat stellate cells, passage 8.

[0117] 3. Human umbilical vein endothelial cells, passage 4 (HUVEC).

[0118] 4. Human aortic adventitial fibroblasts, passage 5 (AoAF).

[0119] 5. Human aortic smooth muscle cells, passage 2 (AoSMC).

[0120] Cells were washed once with ice-cold binding buffer (HAM'S F-12containing 2.5 mg/ml BSA, 20 mM HEPES, pH 7.2), then 250 μl of thefollowing solutions was added to each of three wells of the culturedishes containing the test cells. Binding solutions were prepared in 5mL of binding buffer with 250 pM ¹²⁵I-zvegf4 and:

[0121] 1. No addition.

[0122] 2. 25 nM zvegf4.

[0123] 3. 25 nM zvegf3 (PDGF-C).

[0124] 4. 25 nM PDGF-AA.

[0125] 5. 25 nM PDGF-BB.

[0126] The reaction mixtures were incubated on ice for 2 hours, thenwashed three times with one mil of ice-cold binding buffer. The bound¹²⁵ ^(I)-zvegf4 was quantitated by gamma counting at-octylphenoxypolyethoxyethanol (Triton® X-100) extract of the cells.

[0127] Results, shown in Table 4, indicate binding of zvegf4 topericytes, stellate cells, AoAF, and AoSMC, but not to HUVEC. The firstcolumn represents total CPM ¹²⁵I-zvegf4 bound/well. The second column is¹²⁵I-zvegf4 bound/well when blocked with cold ligand. The differencebetween the two numbers represents specific binding. TABLE 4 ¹²⁵I-zvegf4Bound w/cold Cell Type ¹²⁵I-zvegf4 Bound (CPM) zvegf4 (CPM) 1. Pericytes3083 +/− 864 623 +/− 60 2. Stellate Cells 2131 +/− 450  413 +/− 164 3.HUVEC 485 +/− 91 227 +/− 13 4. AoAF 1544 +/− 131 300 +/− 15 5. AoSMC1628 +/− 203 440 +/− 46

[0128] Zvegf4 binding was not significantly reduced by PDGF-AA orPDGF-BB (data not shown). These results indicate that full-length zvegf4can bind to the tested cells at binding sites distinct from those forthe AA and BB isoforms of PDGF. These zvegf4 binding sites may be either(1) sites on known PDGF receptors that are distinct from the bindingsites for the AA and BB isoforms, or (2) one or more differentmolecules, such as cell-surface semaphorins.

Example 12

[0129] The activity of recombinant human zvegf4 growth factor domain wastested on BHK cell lines that stably expressed the α subunit of the PDGFreceptor, the β subunit of the PDGF receptor, or both the α and βsubunits of the PDGF receptor. Wild-type BHK 570 cells (which do notrespond to PDGF because they do not express adequate levels of PDGFreceptors) and stable BHK cell lines that expressed the human PDGFreceptor subunits were plated at 10-15×10³ cells/well in 96-well cellculture trays in DMEM (Life Technologies, Gaithersburg, Md.)supplemented with 10% fetal bovine serum (HyClone Laboratories, Inc.,Logan, Utah). The medium of the stable clones was further supplementedwith 200 nM methotrexate to maintain stable selection. At 70-80%confluence (the next day), the growth medium was replaced withserum-free medium, and the cells were infected with an adenoviralreporter construct (designated KZ 136) containing a firefly luciferasegene under the control of an SRE-STAT promoter at 1,000:1 multiplicityof infection (1,000 viral particles per cell). Twenty-four hours later,the medium was once more replaced with serum-free medium, andrecombinant human zvegf4 growth factor domain was added to the cells.Four hours later, the cells were lysed, and the luciferase activity inthe lysate was determined using a commercially available kit (obtainedfrom Promega Corporation, Madison, Wis.) and a luminometer device(Luminoskan™; Labsystems Oy, Helsinki, FI) to detect the emittedluminescence. As shown in Tables 5-7, zvegf4 triggered responses in allthree receptor-expressing BHK cell lines (but not in wild-type BHKcells, not shown), indicating that it can signal through αα, ββ and αβPDGF receptor complexes. TABLE 5 BHK expressing the PDGF receptor alphasubunit Zvegf4 Luciferase (ng/ml) Units 0 2.14 0.3 2.12 1 2.92 3 4.50 109.60 30 13.30 100 21.95

[0130] TABLE 6 BHK expressing PDGF receptor beta subunit Zvegf4Luciferase (ng/ml) Units 0 4.49 0.3 8.53 1 14.77 3 20.91 10 37.22 3040.16 100 36.41

[0131] TABLE 7 BHK cells expressing PDGF receptor alpha and betasubunits Zvegf4 Luciferase (ng/ml) Units 0  6.64 0.3 13.99 1 19.11 326.21 10 44.25 30 61.15 100 60.25

Example 13

[0132] Human aortic smooth muscle cells at passage 6 (Clonetics) wereplated at 10×10³ cells/well in 96-well cell culture trays in DMEMsupplemented with 10% fetal bovine serum. At confluence (the next day),the growth medium was replaced with serum-free DMEM containing 0.1% BSA,and the cells were returned to the incubator, allowing for partialgrowth arrest. Twenty-four hours later, the media were once morereplaced with serum-free medium with BSA. Recombinant human zvegf4growth factor domain (30 ng/ml final concentration in the well) wasmixed with neutralizing monoclonal antibodies against the alpha or betaPDGF receptor subunits or with non-immune mouse IgG (20 μg/ml finalconcentration in the well) for 10 minutes at room temperature. Themixture was then added to the cells. [³H]-thymidine at 1 μCi/ml(Amersham, final concentration in the well) was added immediatelyafterwards, and the cells were returned to the incubator for anadditional 24 hours to allow for [³H]-thymidine incorporation into newlysynthesized DNA. The cells were washed twice to remove unincorporatedlabel and were harvested using a sample harvester (FilterMate™ 196harvester; Packard Instrument Co.). Incorporated thymidine wasdetermined using a scintillation counter (Topcount®; Packard InstrumentCo.). Results from triplicate well determinations, expressed as mean ±standard deviation of cpm of radioactivity per well, are shown in Table8. “Response” indicates the fold increase in thymidine incorporationresulting from the addition of zvegf4. The data show that the responseof human aortic smooth muscle cells to zvegf4 was substantially reducedby both anti-alpha and anti-beta PDGF receptor subunit neutralizingantibodies, indicating that both PDGF receptor subunits are bound byzvegf4. TABLE 8 [³H]-Thymidine Antibody zvegf4 (cpm) Response Non-immuneserum (Control) −  81 ± 21 Non-immune serum (Control) + 696 ± 60  8-fold anti-PDGF-Rβ − 116 ± 19 anti-PDGF-Rβ + 151 ± 15 1.4-foldanti-PDGF-Rα − 102 ± 32 anti-PDGF-Rα + 322 ± 64 3.2-fold

Example 14

[0133] Rat stellate cells were grown in 48-well tissue clusters(Falcon™; BD Labware, Bedford, Mass.) in DMEM (Life Technologies, Inc.)supplemented with 10% fetal bovine serum (Hyclone Laboratories, Inc.).At 80% confluence, the cells were switched to growth-arrest medium bysubstituting 0.1% BSA (Sigma-Aldrich Corp., St. Louis, Mo.) for serum.Two days later the growth-arrest medium was replaced with the samemedium, and recombinant human zvegf4 growth factor domain was added tothe cells. After 48 hours, the conditioned media were collected, andTGF-β1 levels were determined using an ELISA kit (obtained from R&DSystems, Minneapolis, Minn.). Results are shown in Table 9. TABLE 9Treatment pg TGF-β1 per well BSA Control  2 ± 3 Zvegf4 3 ng/ml  32 ± 28Zvegf4 30 ng/ml 120 ± 12 Zvegf4 300 ng/ml 175 ± 71

Example 15

[0134] OC10B mouse osteoblasts (Thomson et al., J. Bone Min. Res.,13(2):195-204, 1998) were grown in 96-well tissue clusters (Falcon™)until confluence in DMEM (Life Technologies, Inc.) supplemented with 10%fetal bovine serum (Hyclone Laboratories, Inc.). They were then switchedto growth-arrest medium by substituting 0.1% BSA (Sigma-Aldrich Corp.)for serum. Forty-eight hours later, the growth-arrest medium wasreplaced with the same medium, and recombinant human zvegf4 growthfactor domain was added to the cells. The cells were pulsed with 1μCi/ml [³H]-thymidine (NEN Life Science Products, Inc., Boston, Mass.)for 8 hours, 16-24 hours after addition of zvegf4. The radioactivityincorporated by the cells was determined by harvesting the cells with asample harvester and counting the incorporated label using a microplatescintillation counter.

[0135] Results, shown in Table 10, indicate that zvegf4 directlystimulates osteoblast proliferation. Zvegf4 antagonists may therefore beuseful in reducing growth of osteoblasts, such as in osteosarcomas orosteoblastic prostate metastases. TABLE 10 Treatment cpm/well (10⁻³) BSAControl 17 ± 2  Zvegf4 1 ng/ml 36 ± 11 Zvegf4 3 ng/ml 42 ± 8  Zvegf4 10ng/ml 51 ± 17 Zvegf4 30 ng/ml 59 ± 11 Zvegf4 100 ng/ml 51 ± 25 Zvegf4300 ng/ml 63 ± 13

Example 16

[0136] OC10B cells in vitro can differentiate into both osteoblasts andadipocytes (fat cells) when grown in a medium containing 100 μg/mlascorbic acid and 10 mM beta-glycerophosphate (Thomson et al., ibid.).This differentiation recapitulates the in vivo physiological processwhereby both cell lineages are derived from a common, bi-potentialprogenitor.

[0137] OC10B cells were cultured in the presence of ascorbic acid andbeta-glycerophosphate for 10 days. Zvegf4 suppressed differentiation ofthe cells into adipocytes, as determined by the absence of cellscontaining light-reflective fat droplets. In contrast, there was anincrease in the number and size of mineralized foci as assessed by VonKossa staining.

[0138] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 13 1 1882 DNA Homo sapiens CDS (226)...(1338) 1 ccgtcaccat ttatcagctcagcaccacaa ggaagtgcgg cacccacacg cgctcggaaa 60 gttcagcatg caggaagtttggggagagct cggcgattag cacagcgacc cgggccagcg 120 cagggcgagc gcaggcggcgagagcgcagg gcggcgcggc gtcggtcccg ggagcagaac 180 ccggcttttt cttggagcgacgctgtctct agtcgctgat cccaa atg cac cgg ctc 237 Met His Arg Leu 1 atcttt gtc tac act cta atc tgc gca aac ttt tgc agc tgt cgg gac 285 Ile PheVal Tyr Thr Leu Ile Cys Ala Asn Phe Cys Ser Cys Arg Asp 5 10 15 20 acttct gca acc ccg cag agc gca tcc atc aaa gct ttg cgc aac gcc 333 Thr SerAla Thr Pro Gln Ser Ala Ser Ile Lys Ala Leu Arg Asn Ala 25 30 35 aac ctcagg cga gat gag agc aat cac ctc aca gac ttg tac cga aga 381 Asn Leu ArgArg Asp Glu Ser Asn His Leu Thr Asp Leu Tyr Arg Arg 40 45 50 gat gag accatc cag gtg aaa gga aac ggc tac gtg cag agt cct aga 429 Asp Glu Thr IleGln Val Lys Gly Asn Gly Tyr Val Gln Ser Pro Arg 55 60 65 ttc ccg aac agctac ccc agg aac ctg ctc ctg aca tgg cgg ctt cac 477 Phe Pro Asn Ser TyrPro Arg Asn Leu Leu Leu Thr Trp Arg Leu His 70 75 80 tct cag gag aat acacgg ata cag cta gtg ttt gac aat cag ttt gga 525 Ser Gln Glu Asn Thr ArgIle Gln Leu Val Phe Asp Asn Gln Phe Gly 85 90 95 100 tta gag gaa gca gaaaat gat atc tgt agg tat gat ttt gtg gaa gtt 573 Leu Glu Glu Ala Glu AsnAsp Ile Cys Arg Tyr Asp Phe Val Glu Val 105 110 115 gaa gat ata tcc gaaacc agt acc att att aga gga cga tgg tgt gga 621 Glu Asp Ile Ser Glu ThrSer Thr Ile Ile Arg Gly Arg Trp Cys Gly 120 125 130 cac aag gaa gtt cctcca agg ata aaa tca aga acg aac caa att aaa 669 His Lys Glu Val Pro ProArg Ile Lys Ser Arg Thr Asn Gln Ile Lys 135 140 145 atc aca ttc aag tccgat gac tac ttt gtg gct aaa cct gga ttc aag 717 Ile Thr Phe Lys Ser AspAsp Tyr Phe Val Ala Lys Pro Gly Phe Lys 150 155 160 att tat tat tct ttgctg gaa gat ttc caa ccc gca gca gct tca gag 765 Ile Tyr Tyr Ser Leu LeuGlu Asp Phe Gln Pro Ala Ala Ala Ser Glu 165 170 175 180 acc aac tgg gaatct gtc aca agc tct att tca ggg gta tcc tat aac 813 Thr Asn Trp Glu SerVal Thr Ser Ser Ile Ser Gly Val Ser Tyr Asn 185 190 195 tct cca tca gtaacg gat ccc act ctg att gcg gat gct ctg gac aaa 861 Ser Pro Ser Val ThrAsp Pro Thr Leu Ile Ala Asp Ala Leu Asp Lys 200 205 210 aaa att gca gaattt gat aca gtg gaa gat ctg ctc aag tac ttc aat 909 Lys Ile Ala Glu PheAsp Thr Val Glu Asp Leu Leu Lys Tyr Phe Asn 215 220 225 cca gag tca tggcaa gaa gat ctt gag aat atg tat ctg gac acc cct 957 Pro Glu Ser Trp GlnGlu Asp Leu Glu Asn Met Tyr Leu Asp Thr Pro 230 235 240 cgg tat cga ggcagg tca tac cat gac cgg aag tca aaa gtt gac ctg 1005 Arg Tyr Arg Gly ArgSer Tyr His Asp Arg Lys Ser Lys Val Asp Leu 245 250 255 260 gat agg ctcaat gat gat gcc aag cgt tac agt tgc act ccc agg aat 1053 Asp Arg Leu AsnAsp Asp Ala Lys Arg Tyr Ser Cys Thr Pro Arg Asn 265 270 275 tac tcg gtcaat ata aga gaa gag ctg aag ttg gcc aat gtg gtc ttc 1101 Tyr Ser Val AsnIle Arg Glu Glu Leu Lys Leu Ala Asn Val Val Phe 280 285 290 ttt cca cgttgc ctc ctc gtg cag cgc tgt gga gga aat tgt ggc tgt 1149 Phe Pro Arg CysLeu Leu Val Gln Arg Cys Gly Gly Asn Cys Gly Cys 295 300 305 gga act gtcaac tgg agg tcc tgc aca tgc aat tca ggg aaa acc gtg 1197 Gly Thr Val AsnTrp Arg Ser Cys Thr Cys Asn Ser Gly Lys Thr Val 310 315 320 aaa aag tatcat gag gta tta cag ttt gag cct ggc cac atc aag agg 1245 Lys Lys Tyr HisGlu Val Leu Gln Phe Glu Pro Gly His Ile Lys Arg 325 330 335 340 agg ggtaga gct aag acc atg gct cta gtt gac atc cag ttg gat cac 1293 Arg Gly ArgAla Lys Thr Met Ala Leu Val Asp Ile Gln Leu Asp His 345 350 355 cat gaacga tgc gat tgt atc tgc agc tca aga cca cct cga taa 1338 His Glu Arg CysAsp Cys Ile Cys Ser Ser Arg Pro Pro Arg * 360 365 370 gagaatgtgcacatccttac attaagcctg aaagaacctt tagtttaagg agggtgagat 1398 aagagacccttttcctacca gcaaccaaac ttactactag cctgcaatgc aatgaacaca 1458 agtggttgctgagtctcagc cttgctttgt taatgccatg gcaagtagaa aggtatatca 1518 tcaacttctatacctaagaa tataggattg catttaataa tagtgtttga ggttatatat 1578 gcacaaacacacacagaaat atattcatgt ctatgtgtat atagatcaaa tgtttttttt 1638 ttttggtatatataaccagg tacaccagag gttacatatg tttgagttag actcttaaaa 1698 tcctttgccaaaataaggga tggtcaaata tatgaaacat gtctttagaa aatttaggag 1758 ataaatttatttttaaattt tgaaacacga aacaattttg aatcttgctc tcttaaagaa 1818 agcatcttgtatattaaaaa tcaaaagatg aggctttctt acatatacat cttagttgat 1878 tatt 1882 2370 PRT Homo sapiens 2 Met His Arg Leu Ile Phe Val Tyr Thr Leu Ile CysAla Asn Phe Cys 1 5 10 15 Ser Cys Arg Asp Thr Ser Ala Thr Pro Gln SerAla Ser Ile Lys Ala 20 25 30 Leu Arg Asn Ala Asn Leu Arg Arg Asp Glu SerAsn His Leu Thr Asp 35 40 45 Leu Tyr Arg Arg Asp Glu Thr Ile Gln Val LysGly Asn Gly Tyr Val 50 55 60 Gln Ser Pro Arg Phe Pro Asn Ser Tyr Pro ArgAsn Leu Leu Leu Thr 65 70 75 80 Trp Arg Leu His Ser Gln Glu Asn Thr ArgIle Gln Leu Val Phe Asp 85 90 95 Asn Gln Phe Gly Leu Glu Glu Ala Glu AsnAsp Ile Cys Arg Tyr Asp 100 105 110 Phe Val Glu Val Glu Asp Ile Ser GluThr Ser Thr Ile Ile Arg Gly 115 120 125 Arg Trp Cys Gly His Lys Glu ValPro Pro Arg Ile Lys Ser Arg Thr 130 135 140 Asn Gln Ile Lys Ile Thr PheLys Ser Asp Asp Tyr Phe Val Ala Lys 145 150 155 160 Pro Gly Phe Lys IleTyr Tyr Ser Leu Leu Glu Asp Phe Gln Pro Ala 165 170 175 Ala Ala Ser GluThr Asn Trp Glu Ser Val Thr Ser Ser Ile Ser Gly 180 185 190 Val Ser TyrAsn Ser Pro Ser Val Thr Asp Pro Thr Leu Ile Ala Asp 195 200 205 Ala LeuAsp Lys Lys Ile Ala Glu Phe Asp Thr Val Glu Asp Leu Leu 210 215 220 LysTyr Phe Asn Pro Glu Ser Trp Gln Glu Asp Leu Glu Asn Met Tyr 225 230 235240 Leu Asp Thr Pro Arg Tyr Arg Gly Arg Ser Tyr His Asp Arg Lys Ser 245250 255 Lys Val Asp Leu Asp Arg Leu Asn Asp Asp Ala Lys Arg Tyr Ser Cys260 265 270 Thr Pro Arg Asn Tyr Ser Val Asn Ile Arg Glu Glu Leu Lys LeuAla 275 280 285 Asn Val Val Phe Phe Pro Arg Cys Leu Leu Val Gln Arg CysGly Gly 290 295 300 Asn Cys Gly Cys Gly Thr Val Asn Trp Arg Ser Cys ThrCys Asn Ser 305 310 315 320 Gly Lys Thr Val Lys Lys Tyr His Glu Val LeuGln Phe Glu Pro Gly 325 330 335 His Ile Lys Arg Arg Gly Arg Ala Lys ThrMet Ala Leu Val Asp Ile 340 345 350 Gln Leu Asp His His Glu Arg Cys AspCys Ile Cys Ser Ser Arg Pro 355 360 365 Pro Arg 370 3 1472 DNA Musmusculus CDS (93)...(1205) 3 agggactgtg cagtagaaat ccgccgactc aaccctttgggctttattta tttacttttg 60 gagcaacgcg atccctaggt cgctgagccc aa atg caa cggctc gtt tta gtc 113 Met Gln Arg Leu Val Leu Val 1 5 tcc att ctc ctg tgcgcg aac ttt agc tgc tat ccg gac act ttt gcg 161 Ser Ile Leu Leu Cys AlaAsn Phe Ser Cys Tyr Pro Asp Thr Phe Ala 10 15 20 act ccg cag aga gca tccatc aaa gct ttg cgc aat gcc aac ctc agg 209 Thr Pro Gln Arg Ala Ser IleLys Ala Leu Arg Asn Ala Asn Leu Arg 25 30 35 aga gat gag agc aat cac ctcaca gac ttg tac cag aga gag gag aac 257 Arg Asp Glu Ser Asn His Leu ThrAsp Leu Tyr Gln Arg Glu Glu Asn 40 45 50 55 att cag gtg aca agc aat ggccat gtg cag agt cct cgc ttc ccg aac 305 Ile Gln Val Thr Ser Asn Gly HisVal Gln Ser Pro Arg Phe Pro Asn 60 65 70 agc tac cca agg aac ctg ctt ctgaca tgg tgg ctc cgt tcc cag gag 353 Ser Tyr Pro Arg Asn Leu Leu Leu ThrTrp Trp Leu Arg Ser Gln Glu 75 80 85 aaa aca cgg ata caa ctg tcc ttt gaccat caa ttc gga cta gag gaa 401 Lys Thr Arg Ile Gln Leu Ser Phe Asp HisGln Phe Gly Leu Glu Glu 90 95 100 gca gaa aat gac att tgt agg tat gacttt gtg gaa gtt gaa gaa gtc 449 Ala Glu Asn Asp Ile Cys Arg Tyr Asp PheVal Glu Val Glu Glu Val 105 110 115 tca gag agc agc act gtt gtc aga ggaaga tgg tgt ggc cac aag gag 497 Ser Glu Ser Ser Thr Val Val Arg Gly ArgTrp Cys Gly His Lys Glu 120 125 130 135 atc cct cca agg ata acg tca agaaca aac cag att aaa atc aca ttt 545 Ile Pro Pro Arg Ile Thr Ser Arg ThrAsn Gln Ile Lys Ile Thr Phe 140 145 150 aag tct gat gac tac ttt gtg gcaaaa cct gga ttc aag att tat tat 593 Lys Ser Asp Asp Tyr Phe Val Ala LysPro Gly Phe Lys Ile Tyr Tyr 155 160 165 tca ttt gtg gaa gat ttc caa ccggaa gca gcc tca gag acc aac tgg 641 Ser Phe Val Glu Asp Phe Gln Pro GluAla Ala Ser Glu Thr Asn Trp 170 175 180 gaa tca gtc aca agc tct ttc tctggg gtg tcc tat cac tct cca tca 689 Glu Ser Val Thr Ser Ser Phe Ser GlyVal Ser Tyr His Ser Pro Ser 185 190 195 ata acg gac ccc act ctc act gctgat gcc ctg gac aaa act gtc gca 737 Ile Thr Asp Pro Thr Leu Thr Ala AspAla Leu Asp Lys Thr Val Ala 200 205 210 215 gaa ttc gat acc gtg gaa gatcta ctt aag cac ttc aat cca gtg tct 785 Glu Phe Asp Thr Val Glu Asp LeuLeu Lys His Phe Asn Pro Val Ser 220 225 230 tgg caa gat gat ctg gag aatttg tat ctg gac acc cct cat tat aga 833 Trp Gln Asp Asp Leu Glu Asn LeuTyr Leu Asp Thr Pro His Tyr Arg 235 240 245 ggc agg tca tac cat gat cggaag tcc aaa gtg gac ctg gac agg ctc 881 Gly Arg Ser Tyr His Asp Arg LysSer Lys Val Asp Leu Asp Arg Leu 250 255 260 aat gat gat gtc aag cgt tacagt tgc act ccc agg aat cac tct gtg 929 Asn Asp Asp Val Lys Arg Tyr SerCys Thr Pro Arg Asn His Ser Val 265 270 275 aac ctc agg gag gag ctg aagctg acc aat gca gtc ttc ttc cca cga 977 Asn Leu Arg Glu Glu Leu Lys LeuThr Asn Ala Val Phe Phe Pro Arg 280 285 290 295 tgc ctc ctc gtg cag cgctgt ggt ggc aac tgt ggt tgc gga act gtc 1025 Cys Leu Leu Val Gln Arg CysGly Gly Asn Cys Gly Cys Gly Thr Val 300 305 310 aac tgg aag tcc tgc acatgc agc tca ggg aag aca gtg aag aag tat 1073 Asn Trp Lys Ser Cys Thr CysSer Ser Gly Lys Thr Val Lys Lys Tyr 315 320 325 cat gag gta ttg aag tttgag cct gga cat ttc aag aga agg ggc aaa 1121 His Glu Val Leu Lys Phe GluPro Gly His Phe Lys Arg Arg Gly Lys 330 335 340 gct aag aat atg gct cttgtt gat atc cag ctg gat cat cat gag cga 1169 Ala Lys Asn Met Ala Leu ValAsp Ile Gln Leu Asp His His Glu Arg 345 350 355 tgt gac tgt atc tgc agctca aga cca cct cga taa aacactatgc 1215 Cys Asp Cys Ile Cys Ser Ser ArgPro Pro Arg * 360 365 370 acatctgtac tttgattatg aaaggacctt taggttacaaaaaccctaag aagcttctaa 1275 tctcagtgca atgaatgcat atggaaatgt tgctttgttagtgccatggc aagaagaagc 1335 aaatatcatt aatttctata tacataaaca taggaattcacttatcaata gtatgtgaag 1395 atatgtatat atacttatat acatgactag ctctatgtatgtaaatagat taaatacttt 1455 attcagtata tttactg 1472 4 370 PRT Musmusculus 4 Met Gln Arg Leu Val Leu Val Ser Ile Leu Leu Cys Ala Asn PheSer 1 5 10 15 Cys Tyr Pro Asp Thr Phe Ala Thr Pro Gln Arg Ala Ser IleLys Ala 20 25 30 Leu Arg Asn Ala Asn Leu Arg Arg Asp Glu Ser Asn His LeuThr Asp 35 40 45 Leu Tyr Gln Arg Glu Glu Asn Ile Gln Val Thr Ser Asn GlyHis Val 50 55 60 Gln Ser Pro Arg Phe Pro Asn Ser Tyr Pro Arg Asn Leu LeuLeu Thr 65 70 75 80 Trp Trp Leu Arg Ser Gln Glu Lys Thr Arg Ile Gln LeuSer Phe Asp 85 90 95 His Gln Phe Gly Leu Glu Glu Ala Glu Asn Asp Ile CysArg Tyr Asp 100 105 110 Phe Val Glu Val Glu Glu Val Ser Glu Ser Ser ThrVal Val Arg Gly 115 120 125 Arg Trp Cys Gly His Lys Glu Ile Pro Pro ArgIle Thr Ser Arg Thr 130 135 140 Asn Gln Ile Lys Ile Thr Phe Lys Ser AspAsp Tyr Phe Val Ala Lys 145 150 155 160 Pro Gly Phe Lys Ile Tyr Tyr SerPhe Val Glu Asp Phe Gln Pro Glu 165 170 175 Ala Ala Ser Glu Thr Asn TrpGlu Ser Val Thr Ser Ser Phe Ser Gly 180 185 190 Val Ser Tyr His Ser ProSer Ile Thr Asp Pro Thr Leu Thr Ala Asp 195 200 205 Ala Leu Asp Lys ThrVal Ala Glu Phe Asp Thr Val Glu Asp Leu Leu 210 215 220 Lys His Phe AsnPro Val Ser Trp Gln Asp Asp Leu Glu Asn Leu Tyr 225 230 235 240 Leu AspThr Pro His Tyr Arg Gly Arg Ser Tyr His Asp Arg Lys Ser 245 250 255 LysVal Asp Leu Asp Arg Leu Asn Asp Asp Val Lys Arg Tyr Ser Cys 260 265 270Thr Pro Arg Asn His Ser Val Asn Leu Arg Glu Glu Leu Lys Leu Thr 275 280285 Asn Ala Val Phe Phe Pro Arg Cys Leu Leu Val Gln Arg Cys Gly Gly 290295 300 Asn Cys Gly Cys Gly Thr Val Asn Trp Lys Ser Cys Thr Cys Ser Ser305 310 315 320 Gly Lys Thr Val Lys Lys Tyr His Glu Val Leu Lys Phe GluPro Gly 325 330 335 His Phe Lys Arg Arg Gly Lys Ala Lys Asn Met Ala LeuVal Asp Ile 340 345 350 Gln Leu Asp His His Glu Arg Cys Asp Cys Ile CysSer Ser Arg Pro 355 360 365 Pro Arg 370 5 24 DNA Artificial Sequenceoligonucleotide primer ZC21,987 5 caacctgttg tttgtcccgt cacc 24 6 24 DNAArtificial Sequence oligonucleotide primer ZC21,120 6 tccagagcatccgcaatcag agtg 24 7 25 DNA Artificial Sequence oligonucleotide primerZC26317 7 atcacctcac agacttgtac cagag 25 8 25 DNA Artificial Sequenceoligonucleotide primer ZC26318 8 cctacaaatg tcattttctg cttcc 25 9 18 PRTArtificial Sequence peptide 9 Cys Gly His Lys Glu Val Pro Pro Arg IleLys Ser Arg Thr Asn Gln 1 5 10 15 Ile Lys 10 25 PRT Artificial Sequencepeptide 10 Glu Ser Trp Gln Glu Asp Leu Glu Asn Met Tyr Leu Asp Thr ProArg 1 5 10 15 Tyr Arg Gly Arg Ser Tyr His Asp Cys 20 25 11 24 PRTArtificial Sequence peptide 11 Cys Phe Glu Pro Gly His Ile Lys Arg ArgGly Arg Ala Lys Thr Met 1 5 10 15 Ala Leu Val Asp Ile Gln Leu Asp 20 126 PRT Artificial Sequence peptide 12 Glu Tyr Met Pro Met Glu 1 5 13 6PRT Artificial Sequence peptide 13 Glu Tyr Met Pro Thr Asp 1 5

What is claimed is:
 1. A method of reducing proliferation of orextracellular matrix production by a cell in a mammal comprisingadministering to the mammal a composition comprising a therapeuticallyeffective amount of a zvegf4 antagonist in combination with apharmaceutically acceptable delivery vehicle, wherein the zvegf4antagonist is selected from the group consisting of: anti-zvegf4antibodies; inhibitory polynucleotides; inhibitors of zvegf4 activation;and mitogenically inactive, receptor-binding variants of zvegf4.
 2. Themethod of claim 1 wherein proliferation of mesangial, epithelial,endothelial, smooth muscle, fibroblast, osteoblast, osteoclast,neuronal, stromal, stellate, or interstitial cells is reduced.
 3. Themethod of claim 1 wherein proliferation of tumor cells is reduced. 4.The method of claim 3 wherein the tumor cells are prostate tumor cells.5. The method of claim 1 wherein extracellular matrix production isreduced.
 6. The method of claim 1 wherein the mammal is suffering from afibroproliferative disorder of kidney.
 7. The method of claim 1 whereinthe mammal is suffering from a fibroproliferative disorder of liver. 8.The method of claim 1 wherein the mammal is suffering from afibroproliferative disorder of bone.
 9. The method of claim 1 whereinthe zvegf4 antagonist is selected from the group consisting ofanti-zvegf4 antibodies and inhibitory polynucleotides.
 10. The method ofclaim 9 wherein the antagonist is an anti-zvegf4 antibody.
 11. Themethod of claim 10 wherein the antibody is a monoclonal antibody. 12.The method of claim 9 wherein the antagonist is an inhibitorypolynucleotide selected from the group consisting of antisensepolynucleotides, ribozyme-encoding polynucleotides, and external guidesequence-encoding polynucleotides.
 13. The method of claim 1 wherein thezvegf4 antagonist is administered in combination with an antagonist of asecond growth factor.
 14. The method of claim 11 wherein the secondgrowth factor is EGF, a TGF-β, or an FGF.
 15. A method of reducingproliferation of or extracellular matrix production by a cell in amammal, wherein the cell is an epithelial, endothelial, smooth muscle,fibroblast, osteoblast, neuronal, or stellate cell, the methodcomprising administering to the mammal a composition comprising atherapeutically effective amount of a zvegf4 antagonist in combinationwith a pharmaceutically acceptable delivery vehicle, wherein the zvegf4antagonist is selected from the group consisting of: anti-zvegf4antibodies; inhibitory polynucleotides; inhibitors of zvegf4 activation;and mitogenically inactive, receptor-binding variants of zvegf4.
 16. Amethod of reducing proliferation of or extracellular matrix productionby prostate tumor cells in a mammal, the method comprising administeringto the mammal a composition comprising a therapeutically effectiveamount of a zvegf4 antagonist in combination with a pharmaceuticallyacceptable delivery vehicle, wherein the zvegf4 antagonist is selectedfrom the group consisting of: anti-zvegf4 antibodies; inhibitorypolynucleotides; inhibitors of zvegf4 activation; and mitogenicallyinactive, receptor-binding variants of zvegf4.
 17. A method of reducingmetastasis of prostate cancer cells to bone in a mammal, the methodcomprising administering to the mammal a composition comprising atherapeutically effective amount of a zvegf4 antagonist in combinationwith a pharmaceutically acceptable delivery vehicle, wherein the zvegf4antagonist is selected from the group consisting of: anti-zvegf4antibodies; inhibitory polynucleotides; inhibitors of zvegf4 activation;and mitogenically inactive, receptor-binding variants of zvegf4.
 18. Amethod of treating a fibroproliferative disorder in a mammal comprisingadministering to the mammal a composition comprising a therapeuticallyeffective amount of a zvegf4 antagonist in combination with apharmaceutically acceptable delivery vehicle, wherein the zvegf4antagonist is selected from the group consisting of anti-zvegf4antibodies, inhibitors of zvegf4 activation, mitogenically inactivereceptor-binding zvegf4 variant polypeptides, and inhibitorypolynucleotides.
 19. The method of claim 18 wherein thefibroproliferative disorder is a fibroproliferative disorder of liver.20. The method of claim 18 wherein the fibroproliferative disorder is afibroproliferative disorder of kidney.
 21. The method of claim 18wherein the fibroproliferative disorder is a fibroproliferative disorderof bone.
 22. The method of claim 18 wherein the antagonist is ananti-zvegf4 antibody.
 23. The method of claim 22 wherein the antibody isa monoclonal antibody.
 24. A method of reducing stellate cell activationin a mammal comprising administering to the mammal a compositioncomprising a zvegf4 antagonist in combination with a pharmaceuticallyacceptable delivery vehicle, wherein the zvegf4 antagonist is selectedfrom the group consisting of anti-zvegf4 antibodies, mitogenicallyinactive receptor-binding zvegf4 variant polypeptides, and inhibitorypolynucleotides, in an amount sufficient to reduce stellate cellactivation.